This document summarizes different classes of proteolytic enzymes and their mechanisms of action. There are several classes of proteases including serine, aspartate, cysteine, and metalloproteases. Serine proteases like trypsin and chymotrypsin cleave peptides using a catalytic triad of serine, histidine, and aspartate residues. Aspartate proteases also use acid/base catalysis, while cysteine and metalloproteases employ cysteine and zinc residues respectively in their catalytic mechanisms. Proteases are regulated by activation, localization, and inhibition by endogenous protease inhibitors. Lysosomes contain many hydrolytic enzymes including proteases that degrade macromolecules through autophagy and other
Large family of proteolytic enzymes
All have serine residue at their active site which plays a crucial part in the enzymatic activity.
All cleave peptide bonds, by a similar mechanism of action. They differ in their specificity and regulation.
Serine proteases include:
the pancreatic proteases: trypsin, chymotrypsin and elastase,
various tissue/intracellular proteases such as leukocyte elastase
enzymes of the blood clotting cascade
some enzymes of complement system
Many serine proteases are synthesized as inactive precursors (zymogens) which are activated by proteolysis
Multifunctional enzymes contain two or more distinct catalytic activities located in a single polypeptide chain. Fatty acid synthase is a multifunctional enzyme that synthesizes fatty acids through seven distinct enzymatic activities located on three functional domains. DNA polymerase is another multifunctional enzyme that synthesizes DNA and proofreads for errors through its polymerase and exonuclease activities.
Metabolism of Sulfur Containing Amino Acids (Methionine, Cysteine, Cystine)Ashok Katta
Methionine and cysteine are sulfur-containing amino acids involved in important metabolic pathways.
Methionine is an essential amino acid that is converted to S-adenosylmethionine (SAM), which acts as a methyl group donor in transmethylation reactions. SAM is also regenerated back to methionine. Cysteine is synthesized from methionine and serine via cystathionine. It can be catabolized through transamination or direct oxidation pathways.
Genetic disorders of methionine and cysteine metabolism include cystinuria, cystinosis, hypermethioninemia, and different types of homocystinurias caused by defects in enzymes involved in
Serine proteases are digestive enzymes like trypsin, chymotrypsin, and elastase that differ in substrate specificity. Their catalytic mechanism involves a catalytic triad of serine, histidine, and aspartate residues. During catalysis, the serine hydroxyl performs a nucleophilic attack on the substrate peptide bond, forming a transient acyl-enzyme intermediate before hydrolysis releases the cleaved peptides. Chymotrypsin prefers substrates with aromatic or large hydrophobic residues at the cleavage site.
Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College.
Addition:
There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
This document discusses the metabolism of sulfur-containing amino acids like methionine and cysteine.
It first describes the catabolism of methionine and the formation of cysteine. It then discusses the resynthesis of methionine through remethylation reactions using vitamins B12 and folate as cofactors.
The catabolism of cysteine through transamination and oxidative pathways is also covered. Cysteine serves important roles like participating in protein structure through disulfide bonds and acting as a precursor for glutathione, taurine, and iron-sulfur clusters.
The document concludes by briefly mentioning some metabolic disorders that can arise from defects in sulfur amino acid metabolism like cystinuria, cyst
The citric acid cycle provides precursors for biosynthetic pathways and serves catabolic and anabolic processes. It is regulated by substrate availability and product inhibition. Anaplerotic reactions replenish cycle intermediates used for biosynthesis. Acetyl-CoA derived from the cycle is used in fatty acid synthesis in the cytoplasm. The glyoxylate cycle allows conversion of acetate to carbohydrates in some organisms.
The document summarizes protein degradation. It discusses that protein half-lives vary from minutes to infinity, and enzymes have particularly short half-lives ranging from 11 minutes to 2 hours. The two main pathways of protein degradation are the ubiquitin-proteasome pathway, which degrades the majority of intracellular proteins, and lysosomal degradation, which degrades extracellular and some intracellular proteins. The ubiquitin-proteasome pathway involves ubiquitination of proteins by E1-E3 enzymes, recognition and degradation by the proteasome, and deubiquitination.
Large family of proteolytic enzymes
All have serine residue at their active site which plays a crucial part in the enzymatic activity.
All cleave peptide bonds, by a similar mechanism of action. They differ in their specificity and regulation.
Serine proteases include:
the pancreatic proteases: trypsin, chymotrypsin and elastase,
various tissue/intracellular proteases such as leukocyte elastase
enzymes of the blood clotting cascade
some enzymes of complement system
Many serine proteases are synthesized as inactive precursors (zymogens) which are activated by proteolysis
Multifunctional enzymes contain two or more distinct catalytic activities located in a single polypeptide chain. Fatty acid synthase is a multifunctional enzyme that synthesizes fatty acids through seven distinct enzymatic activities located on three functional domains. DNA polymerase is another multifunctional enzyme that synthesizes DNA and proofreads for errors through its polymerase and exonuclease activities.
Metabolism of Sulfur Containing Amino Acids (Methionine, Cysteine, Cystine)Ashok Katta
Methionine and cysteine are sulfur-containing amino acids involved in important metabolic pathways.
Methionine is an essential amino acid that is converted to S-adenosylmethionine (SAM), which acts as a methyl group donor in transmethylation reactions. SAM is also regenerated back to methionine. Cysteine is synthesized from methionine and serine via cystathionine. It can be catabolized through transamination or direct oxidation pathways.
Genetic disorders of methionine and cysteine metabolism include cystinuria, cystinosis, hypermethioninemia, and different types of homocystinurias caused by defects in enzymes involved in
Serine proteases are digestive enzymes like trypsin, chymotrypsin, and elastase that differ in substrate specificity. Their catalytic mechanism involves a catalytic triad of serine, histidine, and aspartate residues. During catalysis, the serine hydroxyl performs a nucleophilic attack on the substrate peptide bond, forming a transient acyl-enzyme intermediate before hydrolysis releases the cleaved peptides. Chymotrypsin prefers substrates with aromatic or large hydrophobic residues at the cleavage site.
Presentation given by Dr. Karthikeyan at Department of Biochemistry, Maulana Azad Medical College.
Addition:
There are certain proteins which are degraded by proteasome without ubiquitin tag. one such example is ornithine decarboxylase - rate limiting enzyme of polyamine synthesis.
This document discusses the metabolism of sulfur-containing amino acids like methionine and cysteine.
It first describes the catabolism of methionine and the formation of cysteine. It then discusses the resynthesis of methionine through remethylation reactions using vitamins B12 and folate as cofactors.
The catabolism of cysteine through transamination and oxidative pathways is also covered. Cysteine serves important roles like participating in protein structure through disulfide bonds and acting as a precursor for glutathione, taurine, and iron-sulfur clusters.
The document concludes by briefly mentioning some metabolic disorders that can arise from defects in sulfur amino acid metabolism like cystinuria, cyst
The citric acid cycle provides precursors for biosynthetic pathways and serves catabolic and anabolic processes. It is regulated by substrate availability and product inhibition. Anaplerotic reactions replenish cycle intermediates used for biosynthesis. Acetyl-CoA derived from the cycle is used in fatty acid synthesis in the cytoplasm. The glyoxylate cycle allows conversion of acetate to carbohydrates in some organisms.
The document summarizes protein degradation. It discusses that protein half-lives vary from minutes to infinity, and enzymes have particularly short half-lives ranging from 11 minutes to 2 hours. The two main pathways of protein degradation are the ubiquitin-proteasome pathway, which degrades the majority of intracellular proteins, and lysosomal degradation, which degrades extracellular and some intracellular proteins. The ubiquitin-proteasome pathway involves ubiquitination of proteins by E1-E3 enzymes, recognition and degradation by the proteasome, and deubiquitination.
Protease Enzyme Application in Food Processing Mohan Naik
This document discusses proteases, which are enzymes that break down proteins. It notes that proteases are widely distributed in biological systems and constitute over 70% of industrial enzymes. Proteases have a wide range of applications including in detergents, food processing, pharmaceuticals, and leather tanning. The document categorizes proteases based on their source (animal, plant, bacterial, fungal), proteolytic mechanism (serine, threonine, cysteine, aspartic, metallo), and pH range (acidic, neutral, alkaline). It provides examples of major industrial uses of proteases in bread making, cheese production, and soy sauce manufacturing. Protease applications also include meat tenderizing, medicine
Proteoglycans are protein chains that are covalently bonded at multiple sites to a class of polysaccharides, known as glycosaminoglycans.Glycosaminoglycans constitute 95% of proteins.Proteoglycans are synthesised in RE and transported to GA where they are modified in to various forms.Proteoglycans are major component of ECM and their role is depended on the type of GAGs they associate with.
As an essential amino acid, methionine is not synthesized de novo in humans and other animals, which must ingest methionine or methionine-containing proteins. In plants and microorganisms, methionine biosynthesis belongs to the aspartate family, along with threonine and lysine (via diaminopimelate, but not via α-aminoadipate). The main backbone is derived from aspartic acid, while the sulfur may come from cysteine, methanethiol, or hydrogen sulfide.
This document outlines key hormones that regulate metabolic homeostasis, including insulin, glucagon, epinephrine, and cortisol. It describes their structure, biosynthesis, mechanisms of action, and metabolic effects. Insulin promotes anabolism and lowers blood glucose levels, while glucagon, epinephrine, and cortisol have catabolic effects and increase blood glucose as counterregulatory hormones opposed to insulin. Precise regulation of these hormones maintains stable blood glucose levels and fuels metabolism.
Metabolism of Basic Amino Acids (Arginine, Histidine, Lysine)Ashok Katta
This document summarizes amino acid metabolism, including the synthesis and degradation pathways of arginine, histidine, lysine, and their importance. It discusses how arginine is involved in nitric oxide synthesis and polyamine synthesis. Histidine degradation produces histamine. Lysine is involved in carnitine synthesis. Disorders are discussed for each amino acid pathway.
This document discusses bisubstrate reactions, which involve two substrates and produce two products. Approximately 60% of biochemical reactions are bisubstrate reactions. They can be transferase reactions, where a functional group is transferred between substrates, or oxidation-reduction reactions. Bisubstrate reactions fall into two categories: sequential reactions, where substrates bind sequentially before products are released; and ping pong reactions, where an intermediate enzyme form is produced after the first substrate reaction. Common examples of each type of bisubstrate reaction are provided.
Mechanism of action of Chymotrypsin & Lysozyme.pptxVanshikaVarshney5
Chymotrypsin and Lysozyme are the most important enzymes. Mechanism of action of these enzymes and introduction of these enzyme are given in this presentation in simple, easy and understanding language. Hope you will find it useful :)
Laboratory method for measuring enzyme activity.
Vital for study of enzyme kinetics and enzyme inhibition.
Measurement of enzyme activity – follow the change in concentration of substrate or product – measure reaction rate.
Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides through reduction of the 2'-OH group on the ribose sugar. It is a heterotetrameric enzyme composed of R1 and R2 subunits, with the R2 subunit containing a tyrosyl free radical required for activity. Thioredoxin provides reducing equivalents to ribonucleotide reductase through thioredoxin reductase and NADPH to allow for continuous reduction of ribonucleotides to the corresponding deoxyribonucleotides needed for DNA synthesis.
- Methionine and cysteine are sulfur-containing amino acids. Methionine is an essential amino acid while cysteine can be synthesized from methionine and serine.
- There are three major metabolic routes for methionine and cysteine: 1) methionine is used for transmethylation, 2) methionine is used for cysteine synthesis, and 3) cysteine is broken down to make specialized products.
- Deficiencies in enzymes involved in methionine and cysteine metabolism can cause inborn errors such as homocystinuria, cystathioninuria, and cystinosis.
Proteoglycans are complex macromolecules consisting of a core protein with one or more glycosaminoglycan chains attached. They are found mainly in connective tissues and help modulate cellular development processes. Glycoproteins contain oligosaccharide chains covalently bonded to amino acids on their polypeptide side chains. They are found in cellular membranes and function in cellular recognition. Some examples of glycoproteins discussed are mucins, transferrins, fibrinogen, follicle-stimulating hormone, and erythropoietin.
Thiamine pyrophosphate (TPP) is an important coenzyme that maintains normal heart and energy metabolism functions. TPP works as a coenzyme in several enzymatic reactions including pyruvate decarboxylase, transketolase, pyruvate dehydrogenase, and alpha ketoglutarate dehydrogenase. As a cofactor for pyruvate decarboxylase, TPP facilitates the decarboxylation of pyruvate to acetaldehyde. For transketolase, TPP transfers a two-carbon unit from xylulose 5-phosphate to ribose 5-phosphate, yielding sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate. The mechanisms of these reactions
Proteases are enzymes that break down proteins into smaller polypeptides or amino acids through proteolysis. They serve several important functions including protein digestion, blood coagulation, cell growth and apoptosis, immune response, and wound healing. Proteases can be derived from plants, microorganisms, and animals and are classified based on their reaction type, site of enzyme action, pH level, and catalytic mechanism. They have applications in industries like food processing, detergents, pharmaceuticals, and waste management.
Enzymes are biological catalysts that speed up chemical reactions without being consumed. Their activity can be measured by determining the amount of substrate converted to product per unit time. There are two main types of enzyme assays: continuous assays that measure reaction rates over time, and discontinuous assays that take samples at intervals to measure substrate/product levels. Common techniques to measure enzyme activity include spectrophotometry, fluorescence spectroscopy, chromatography, and radiometric methods. Spectrophotometry is often used to examine light absorption of substrates and products in the ultraviolet-visible range.
post translational modifications of proteinAnandhan Ctry
Post-translational modifications (PTMs) are chemical modifications of proteins that occur after translation. PTMs play a key role in regulating protein function by modifying activity, localization, and interactions. The main types of PTMs discussed are phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis. These modifications are identified through techniques like mass spectrometry, HPLC, radioactive labeling, and gel electrophoresis. PTMs are important for processes like cell signaling, growth, and apoptosis.
The document discusses several models of enzyme action and specificity. It describes the lock and key model and induced fit model, where the active site either pre-exists in a rigid confirmation or flexibly changes shape upon substrate binding. It also discusses how substrate binding can induce a change in substrate structure to promote reaction. Finally, it outlines different types of enzyme specificity, including stereo, reaction, and substrate specificity. Enzymes precisely recognize and act only on specific substrates or stereoisomers.
this will be useful to understand about the new topics such as abzymes, ribozymes and also isoenzymes. You have to clear that ribozymes are not protein. because all enzymes are proteins but all proteins are not enzymes except ribozymes
Methods of enzyme isolation and purificationAkshay Wakte
Enzymes are important biological molecules found in living systems. Early attempts at purifying enzymes were made in the 1920s. Methods are needed to isolate enzymes for further study and applications. Common methods to isolate enzymes include breaking open cell walls through grinding, freezing and thawing, using hydrolytic enzymes, blending, altering pH or ionic strength, and using organic solvents. Further purification techniques include centrifugation, gel filtration chromatography, affinity chromatography, and changing solubility through pH or salt concentration. Specific methods have also been developed for isolating individual enzymes like urease and pepsin.
Proteolysis, protein degradation and turnoveremicica
This document discusses protein degradation and turnover. It begins by introducing proteolysis, which is the cleavage of peptide bonds by proteases. Limited proteolysis can create new protein products with new functions through posttranslational modification. There are two main types of proteases - exopeptidases that cleave near the ends of proteins, and endopeptidases that cleave internally. Protein degradation involves complete breakdown of proteins into amino acids by multiple cleavages and occurs through two main pathways: lysosomal breakdown in acidic lysosomes using individual proteases, and ubiquitin-dependent breakdown by the proteasome in the cytosol at neutral pH.
Methods of Gene Transfer document discusses various methods of transferring genes into plants to create transgenic plants. It describes two main categories of gene transfer methods - physical and biological. Physical methods include microinjection, biolistics (gene gun), electroporation, and particle bombardment. Biological methods include Agrobacterium-mediated transformation, which involves using the bacteria Agrobacterium tumefaciens to transfer DNA into plant cells. The document also discusses transformation cassettes, selection of transgenic plants, analysis of transgenic plants, and some examples of commercially important transgenic crops like golden rice and Roundup Ready corn.
Protease Enzyme Application in Food Processing Mohan Naik
This document discusses proteases, which are enzymes that break down proteins. It notes that proteases are widely distributed in biological systems and constitute over 70% of industrial enzymes. Proteases have a wide range of applications including in detergents, food processing, pharmaceuticals, and leather tanning. The document categorizes proteases based on their source (animal, plant, bacterial, fungal), proteolytic mechanism (serine, threonine, cysteine, aspartic, metallo), and pH range (acidic, neutral, alkaline). It provides examples of major industrial uses of proteases in bread making, cheese production, and soy sauce manufacturing. Protease applications also include meat tenderizing, medicine
Proteoglycans are protein chains that are covalently bonded at multiple sites to a class of polysaccharides, known as glycosaminoglycans.Glycosaminoglycans constitute 95% of proteins.Proteoglycans are synthesised in RE and transported to GA where they are modified in to various forms.Proteoglycans are major component of ECM and their role is depended on the type of GAGs they associate with.
As an essential amino acid, methionine is not synthesized de novo in humans and other animals, which must ingest methionine or methionine-containing proteins. In plants and microorganisms, methionine biosynthesis belongs to the aspartate family, along with threonine and lysine (via diaminopimelate, but not via α-aminoadipate). The main backbone is derived from aspartic acid, while the sulfur may come from cysteine, methanethiol, or hydrogen sulfide.
This document outlines key hormones that regulate metabolic homeostasis, including insulin, glucagon, epinephrine, and cortisol. It describes their structure, biosynthesis, mechanisms of action, and metabolic effects. Insulin promotes anabolism and lowers blood glucose levels, while glucagon, epinephrine, and cortisol have catabolic effects and increase blood glucose as counterregulatory hormones opposed to insulin. Precise regulation of these hormones maintains stable blood glucose levels and fuels metabolism.
Metabolism of Basic Amino Acids (Arginine, Histidine, Lysine)Ashok Katta
This document summarizes amino acid metabolism, including the synthesis and degradation pathways of arginine, histidine, lysine, and their importance. It discusses how arginine is involved in nitric oxide synthesis and polyamine synthesis. Histidine degradation produces histamine. Lysine is involved in carnitine synthesis. Disorders are discussed for each amino acid pathway.
This document discusses bisubstrate reactions, which involve two substrates and produce two products. Approximately 60% of biochemical reactions are bisubstrate reactions. They can be transferase reactions, where a functional group is transferred between substrates, or oxidation-reduction reactions. Bisubstrate reactions fall into two categories: sequential reactions, where substrates bind sequentially before products are released; and ping pong reactions, where an intermediate enzyme form is produced after the first substrate reaction. Common examples of each type of bisubstrate reaction are provided.
Mechanism of action of Chymotrypsin & Lysozyme.pptxVanshikaVarshney5
Chymotrypsin and Lysozyme are the most important enzymes. Mechanism of action of these enzymes and introduction of these enzyme are given in this presentation in simple, easy and understanding language. Hope you will find it useful :)
Laboratory method for measuring enzyme activity.
Vital for study of enzyme kinetics and enzyme inhibition.
Measurement of enzyme activity – follow the change in concentration of substrate or product – measure reaction rate.
Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides through reduction of the 2'-OH group on the ribose sugar. It is a heterotetrameric enzyme composed of R1 and R2 subunits, with the R2 subunit containing a tyrosyl free radical required for activity. Thioredoxin provides reducing equivalents to ribonucleotide reductase through thioredoxin reductase and NADPH to allow for continuous reduction of ribonucleotides to the corresponding deoxyribonucleotides needed for DNA synthesis.
- Methionine and cysteine are sulfur-containing amino acids. Methionine is an essential amino acid while cysteine can be synthesized from methionine and serine.
- There are three major metabolic routes for methionine and cysteine: 1) methionine is used for transmethylation, 2) methionine is used for cysteine synthesis, and 3) cysteine is broken down to make specialized products.
- Deficiencies in enzymes involved in methionine and cysteine metabolism can cause inborn errors such as homocystinuria, cystathioninuria, and cystinosis.
Proteoglycans are complex macromolecules consisting of a core protein with one or more glycosaminoglycan chains attached. They are found mainly in connective tissues and help modulate cellular development processes. Glycoproteins contain oligosaccharide chains covalently bonded to amino acids on their polypeptide side chains. They are found in cellular membranes and function in cellular recognition. Some examples of glycoproteins discussed are mucins, transferrins, fibrinogen, follicle-stimulating hormone, and erythropoietin.
Thiamine pyrophosphate (TPP) is an important coenzyme that maintains normal heart and energy metabolism functions. TPP works as a coenzyme in several enzymatic reactions including pyruvate decarboxylase, transketolase, pyruvate dehydrogenase, and alpha ketoglutarate dehydrogenase. As a cofactor for pyruvate decarboxylase, TPP facilitates the decarboxylation of pyruvate to acetaldehyde. For transketolase, TPP transfers a two-carbon unit from xylulose 5-phosphate to ribose 5-phosphate, yielding sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate. The mechanisms of these reactions
Proteases are enzymes that break down proteins into smaller polypeptides or amino acids through proteolysis. They serve several important functions including protein digestion, blood coagulation, cell growth and apoptosis, immune response, and wound healing. Proteases can be derived from plants, microorganisms, and animals and are classified based on their reaction type, site of enzyme action, pH level, and catalytic mechanism. They have applications in industries like food processing, detergents, pharmaceuticals, and waste management.
Enzymes are biological catalysts that speed up chemical reactions without being consumed. Their activity can be measured by determining the amount of substrate converted to product per unit time. There are two main types of enzyme assays: continuous assays that measure reaction rates over time, and discontinuous assays that take samples at intervals to measure substrate/product levels. Common techniques to measure enzyme activity include spectrophotometry, fluorescence spectroscopy, chromatography, and radiometric methods. Spectrophotometry is often used to examine light absorption of substrates and products in the ultraviolet-visible range.
post translational modifications of proteinAnandhan Ctry
Post-translational modifications (PTMs) are chemical modifications of proteins that occur after translation. PTMs play a key role in regulating protein function by modifying activity, localization, and interactions. The main types of PTMs discussed are phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and proteolysis. These modifications are identified through techniques like mass spectrometry, HPLC, radioactive labeling, and gel electrophoresis. PTMs are important for processes like cell signaling, growth, and apoptosis.
The document discusses several models of enzyme action and specificity. It describes the lock and key model and induced fit model, where the active site either pre-exists in a rigid confirmation or flexibly changes shape upon substrate binding. It also discusses how substrate binding can induce a change in substrate structure to promote reaction. Finally, it outlines different types of enzyme specificity, including stereo, reaction, and substrate specificity. Enzymes precisely recognize and act only on specific substrates or stereoisomers.
this will be useful to understand about the new topics such as abzymes, ribozymes and also isoenzymes. You have to clear that ribozymes are not protein. because all enzymes are proteins but all proteins are not enzymes except ribozymes
Methods of enzyme isolation and purificationAkshay Wakte
Enzymes are important biological molecules found in living systems. Early attempts at purifying enzymes were made in the 1920s. Methods are needed to isolate enzymes for further study and applications. Common methods to isolate enzymes include breaking open cell walls through grinding, freezing and thawing, using hydrolytic enzymes, blending, altering pH or ionic strength, and using organic solvents. Further purification techniques include centrifugation, gel filtration chromatography, affinity chromatography, and changing solubility through pH or salt concentration. Specific methods have also been developed for isolating individual enzymes like urease and pepsin.
Proteolysis, protein degradation and turnoveremicica
This document discusses protein degradation and turnover. It begins by introducing proteolysis, which is the cleavage of peptide bonds by proteases. Limited proteolysis can create new protein products with new functions through posttranslational modification. There are two main types of proteases - exopeptidases that cleave near the ends of proteins, and endopeptidases that cleave internally. Protein degradation involves complete breakdown of proteins into amino acids by multiple cleavages and occurs through two main pathways: lysosomal breakdown in acidic lysosomes using individual proteases, and ubiquitin-dependent breakdown by the proteasome in the cytosol at neutral pH.
Methods of Gene Transfer document discusses various methods of transferring genes into plants to create transgenic plants. It describes two main categories of gene transfer methods - physical and biological. Physical methods include microinjection, biolistics (gene gun), electroporation, and particle bombardment. Biological methods include Agrobacterium-mediated transformation, which involves using the bacteria Agrobacterium tumefaciens to transfer DNA into plant cells. The document also discusses transformation cassettes, selection of transgenic plants, analysis of transgenic plants, and some examples of commercially important transgenic crops like golden rice and Roundup Ready corn.
This document discusses various proteolytic enzymes, including their sources and applications. It describes proteases such as papain from papaya, used as a meat tenderizer, and bromelain from pineapple, also used for meat tenderizing. Pepsin from the stomach aids protein digestion. Rennin produces curdling in milk. Trypsin and cathepsins break down proteins, with trypsin used in cell culture and proteomics. These enzymes degrade proteins through hydrolysis of peptide bonds.
Proteases are enzymes that catalyze the hydrolysis of peptide bonds. They perform proteolysis, the breakdown of proteins into amino acids. There are several classes of proteases that differ in their catalytic mechanism and amino acid specificity, including serine, cysteine, aspartyl, metallo, and threonine proteases. Proteases serve many critical functions, such as protein maturation, intracellular protein degradation, regulating protein activity and localization, blood coagulation, digestion, and apoptosis. They are involved in essential biological processes and are highly regulated due to their irreversible effects on protein substrates.
1. Serine proteases use a catalytic triad of serine, histidine, and aspartate residues to hydrolyze peptide bonds through a nucleophilic attack by the serine residue.
2. Site-directed mutagenesis experiments have demonstrated the importance of these catalytic residues and the oxyanion hole for stabilizing the reaction intermediate. Mutating these residues reduces catalytic activity by several orders of magnitude.
3. Recent evidence suggests additional mechanisms such as low barrier hydrogen bonds and substrate assisted catalysis may contribute to the efficiency of serine protease catalysis.
The document summarizes the process of protein synthesis in three main steps: transcription, translation, and termination. During transcription, RNA polymerase makes an mRNA copy of a DNA sequence. Translation then uses the mRNA to assemble a polypeptide chain via tRNAs and ribosomes. Termination occurs when a stop codon signals the release of the completed protein chain. The central dogma of biology is demonstrated as DNA is transcribed to mRNA which is then translated to protein.
The document discusses the structure and formation of proteins. It explains that amino acids join through condensation reactions to form peptide bonds, releasing a water molecule. Multiple peptide bonds form polypeptide chains of varying lengths that fold into unique 3D shapes to serve as functional proteins, such as structural, storage, transport, hormonal, receptor, contractile, defensive, and enzymatic proteins. Common food sources of protein are also mentioned.
Proteins are composed of amino acids and play many essential roles in the body. They have four levels of structure: primary, secondary, tertiary, and quaternary. The primary structure is the amino acid sequence, secondary involves hydrogen bonding into shapes like alpha helices and beta sheets, tertiary is the 3D folding of these structures, and quaternary involves the assembly of multiple protein subunits. Proteins serve as enzymes, hormones, antibodies, and structures. They undergo synthesis from amino acids and breakdown through catabolism. Disorders can occur if amino acid metabolism is disrupted.
Gene transfer technologies can be used to treat diseases by inserting therapeutic genes into cells. There are viral and non-viral methods of gene transfer. Viral methods use viruses like retroviruses, adenoviruses, and adeno-associated viruses to efficiently deliver genes. Non-viral methods include mechanical techniques like electroporation, microinjection, and biolistics (gene gun), as well as chemical methods like liposomes, calcium phosphate, and polyethylene glycol. Each method has advantages and limitations for different applications in research and potential gene therapy.
This document discusses the ubiquitin-proteasome pathway and its role in plant development, with a focus on E3 ubiquitin ligases. It summarizes that the discovery of the ubiquitin-proteasome pathway was relatively recent. A growing number of E3 ligases have been implicated in various plant developmental processes, such as embryogenesis, hormone signaling, and senescence. Given the large number of E3 ligases encoded in the Arabidopsis genome, many plant developmental biologists are studying individual E3 ligases and their roles.
The document discusses the role of ubiquitin in protein degradation and signal transduction. Ubiquitin is a highly conserved 76 amino acid protein that is used for post-translational modification of other proteins through polyubiquitination or monoubiquitination. Polyubiquitinated proteins are targeted for destruction by the 26S proteasome, while monoubiquitination and different types of polyubiquitination are involved in processes like signal transduction, endocytosis, and DNA repair. The ubiquitination pathway involves E1, E2, and E3 enzymes and deubiquitinating enzymes. Defects in the ubiquitination pathway are associated with diseases like cancer, Alzheimer's, and Parkinson
This document summarizes information about the enzyme lysozyme. It is a 129 amino acid enzyme (EC 3.2.1.17) that catalyzes the breakdown of bacterial cell walls. Lysozyme is naturally found in egg whites, tears, and saliva where it acts as a mild antibiotic. Historically, its antibacterial properties were first observed in 1909 and 1922 and its structure was the first enzyme solved using X-ray crystallography in 1975. Lysozyme is widely distributed in animal tissues and fluids where it helps defend against bacterial infections.
Honors - Cells, insulin, signaling and membranes 1213Michael Edgar
The document discusses cell membranes and transport, including definitions of osmosis and diagrams demonstrating how water moves across selectively permeable membranes depending on solute concentration. It also contains information about the sodium-potassium pump, which actively transports sodium and potassium ions across the plasma membrane against their concentration gradients in order to maintain proper electrolyte balance in cells.
Characterizing Protein Families of Unknown FunctionMorgan Langille
by Morgan G. I. Langille & Jonathan A. Eisen. This scientific poster was presented at the 18th Annual International Meeting on Microbial Genomics at Lake Arrowhead, California, USA. Sept. 12-16, 2010.
1) The document summarizes a study examining the regulation of MT1-MMP in mechanically stimulated bioengineered articular cartilage.
2) The study found that mechanical stimulation upregulates the transcription factor Egr-1, which binds to the MT1-MMP promoter and increases MT1-MMP expression.
3) Blocking Egr-1 binding or MT1-MMP expression prevented mechanical stimulation from increasing extracellular matrix production.
The document lists various protein domains found in different organisms along with their lengths. It includes domains such as cadherin, glucosyl hydrolase, integrin, disulfide oxidoreductase, fasciclin, laminin, ferredoxin reductase, nidogen, von Willebrand factor, carboxipeptidase, hydrolase, invasin, serralysin peptidase, adhesion-like domain, and Zn-metalloprotease. The domains are associated with proteins from different bacteria with lengths ranging from 250 to 3816 amino acids.
Stability studies of proteins and peptides.SULABH910
This document discusses stability studies of proteins and peptides. It covers both chemical and physical degradation mechanisms and factors that influence degradation rates. Chemical degradation includes deamidation, racemization, hydrolysis, disulfide formation, oxidation, and cross-linking. Physical degradation involves changes in structure like denaturation, aggregation, adsorption, and precipitation. Degradation rates depend on factors like pH, temperature, moisture content, and excipients. Kinetics are often first-order and follow Arrhenius behavior, allowing prediction of long-term stability from accelerated studies. Understanding degradation mechanisms is key to developing stable protein and peptide drug formulations.
A gene is selected for protein production and RNA polymerase unwinds the DNA at that gene. It uses one DNA strand as a template to produce mRNA through transcription. The primary mRNA transcript is processed to remove introns, leaving exons that are spliced together. The mature mRNA then binds to ribosomes for translation, where tRNA matches its anticodon to mRNA codons and carries the corresponding amino acids. The ribosome catalyzes peptide bond formation between amino acids specified by mRNA to produce the protein.
1) Gene expression in prokaryotes and eukaryotes is regulated in response to environmental changes through various mechanisms at the transcriptional and post-transcriptional levels.
2) In bacteria, operons control transcription of clusters of genes in response to stimuli like small molecules. Repressible and inducible operons use allosteric effectors to turn transcription on or off.
3) In eukaryotes, gene expression is controlled through chromatin modifications, transcription factors, RNA processing, and noncoding RNAs that regulate mRNA translation and chromatin structure. Cancer results from genetic changes affecting cell cycle control genes.
This document summarizes different classes of proteolytic enzymes (proteases) including serine, cysteine, aspartic acid, and metallo proteases. It describes their catalytic mechanisms and examples of each class. Key points are that serine proteases use a catalytic triad of serine, histidine, and aspartate residues in their mechanisms, while cysteine proteases employ a catalytic cysteine and histidine. Aspartic and metallo proteases also have distinct catalytic residues and mechanisms. The roles of proteases in digestion, lysosomes, and other cellular processes are overviewed.
I apologize, as an AI assistant I do not actually use or perform laboratory work. I do not have personal experience working with different classes of proteases.
Unit III covers enzymes, proteases, carbonic anhydrase, restriction enzymes, and nucleoside monophosphate kinases. It then discusses photosynthesis and biological energy production. Enzymes are proteins that act as catalysts to accelerate reactions without being permanently changed. Proteases are enzymes that cleave proteins. Carbonic anhydrase catalyzes the reversible reaction between carbon dioxide and bicarbonate. Restriction enzymes recognize and cut DNA at specific sequences. Nucleoside monophosphate kinases phosphorylate purine monophosphates as part of converting them to their triphosphate forms for use in DNA synthesis.
Post-translational modifications are important biochemical mechanisms that regulate protein function. Common types of post-translational modifications include phosphorylation, hydroxylation, glycosylation, and methylation. These modifications occur on amino acid side chains or termini and are catalyzed by specific enzymes. For example, phosphorylation regulates enzyme activity, while hydroxylation and glycosylation of amino acids are required for collagen assembly and function. Overall, post-translational modifications expand the functional diversity of the proteome.
Proteases can be classified into four main types - serine, cysteine, aspartic, and metallo proteases. Serine proteases contain a catalytic serine residue and include subtilisins. Cysteine proteases contain a catalytic cysteine-histidine dyad and include papain. Metalloproteases require a divalent metal ion like zinc and include thermolysin. The document discusses the classification, sources, and applications of various protease enzymes.
Chemistry of prostaglandins, leukotrienes and thromboxanesAbhimanyu Awasthi
The document summarizes a presentation on the chemistry of prostaglandins, leukotrienes, and thromboxanes. Prostaglandins, leukotrienes, and thromboxanes are oxygen metabolites of arachidonic acid that form a family of lipid substances with intrinsic biological activities. They are involved in processes like inflammation, platelet aggregation, and vascular homeostasis. The presentation covers their biosynthesis from arachidonic acid, sub-families, properties, and biologically important examples like prostacyclin, thromboxane A2, and leukotriene B4. It also discusses the enzymes and pathways involved in their synthesis.
This document provides an overview of post-translational events. It discusses various post-translational modifications including protein folding, proteolytic cleavage, and chemical modifications such as phosphorylation, acetylation, glycosylation, lipidation, and ubiquitination. These modifications influence the structure, stability, activity, and interactions of proteins and play an important role in cellular functions and signaling pathways. The document also examines specific post-translational modifications in depth, including the processes of protein folding, proteolytic cleavage, and various chemical modifications of proteins.
This document discusses various mechanisms of enzyme regulation in living systems. It begins by explaining that hundreds of enzyme-catalyzed reactions must be precisely controlled for proper cellular functioning. It then describes several key mechanisms by which this regulation can be achieved, including allosteric regulation, isoenzyme expression, zymogen activation, and covalent modification via phosphorylation or glycosylation. Specific examples are provided for each type of regulation, such as feedback inhibition of threonine dehydratase and phosphorylation control of glycogen phosphorylase activity. The document concludes by emphasizing that multiple regulatory strategies acting together ensure survival of the cell and maintenance of homeostasis.
This document discusses lysosomes and chaperone-mediated autophagy (CMA). It provides background on the discovery of lysosomes and their structure and functions. Lysosomes contain hydrolytic enzymes and digest macromolecules, cellular debris, and foreign material. CMA selectively degrades cytosolic proteins through binding to a chaperone and lysosomal membrane protein LAMP-2A. Disruption of CMA is implicated in diseases like Parkinson's and cancer. CMA activity declines with age.
This document provides information about enzymes, including their chemistry, structure, cofactors, mechanism of action, kinetics, and classification. It discusses that enzymes are proteins that act as biological catalysts, speeding up biochemical reactions. They have an active site that binds to substrates. Cofactors such as metal ions and organic molecules are required for some enzyme activities. The mechanism of action involves lowering the activation energy of reactions. Enzyme kinetics examines how factors like temperature, pH, and substrate concentration influence reaction rates. Enzymes are classified based on the type of reaction they catalyze.
Organelles interact to carry out important cellular processes like material uptake/release, protein synthesis, and digestion.
(1) Endocytosis and exocytosis allow cells to take in and release material through vesicle trafficking between the plasma membrane and endosomes/lysosomes. (2) The ER and Golgi apparatus work together to synthesize, modify, and transport proteins, with the ER producing proteins and the Golgi processing and packaging them. (3) Lysosomes digest material through phagocytosis, autophagy, and other pathways, while the proteasome degrades unwanted cytosolic proteins. Defects in these organelle interactions can cause diseases.
Physiological And Pathological Systems Within The...Deb Birch
Redox signalling is an important electron transfer process that regulates many physiological and pathological systems in the circulatory system. It is usually induced by reactive oxygen species and can alter cell processes. Redox signalling involves thiol-based redox couples that regulate imbalances in redox potentials and are linked to changes in redox potentials. Cysteine is an amino acid that can undergo oxidative modifications to perform different functions as part of redox signalling.
V.JAGAN MOHAN RAO is an Assistant Professor at NIPER-KOLKATA and MIPER-KURNOOL who provides information on enzyme chemistry. The document discusses enzyme structure, including active sites and cofactors. It also covers enzyme classification, mechanisms of action such as covalent catalysis and acid-base catalysis, kinetics including factors affecting reaction rates like temperature and pH, and inhibition and activation of enzymes.
This document provides an overview of enzymes, including their chemistry, classification, mechanisms of action, kinetics, inhibition, and activation. It begins with the basic introduction that enzymes are protein catalysts that speed up biochemical reactions. It then covers enzyme structure and components like cofactors. The major sections explain classification of enzymes based on reaction type, mechanisms like induced fit and catalytic types, kinetics concepts like Michaelis-Menten modeling and factors affecting reaction rates, and types of inhibition like competitive and noncompetitive. The document aims to comprehensively summarize the key topics relating to enzymes.
This document provides an overview of enzymes, including their structure, function, classification, and kinetics. Some key points:
- Enzymes are biological catalysts that speed up biochemical reactions. They are typically globular proteins that contain an active site for substrate binding.
- Enzymes are classified based on the type of reaction they catalyze, with the major classes being oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.
- Enzyme kinetics examines how factors like temperature, pH, and substrate concentration influence the rate of enzyme-catalyzed reactions. Enzymes lower the activation energy needed for reactions, speeding them up.
- DNA is made up of nucleotides containing nitrogenous bases, sugars, and phosphates. It takes the form of a double helix with the bases pairing across the strands.
- DNA carries genetic information and can be replicated using its base-pairing properties. It is found within chromosomes in the cell nucleus.
- Chromosomes package long DNA molecules into a compact structure using associated proteins like histones. This allows the DNA to fit within the nucleus.
Enzymes are biological molecules (proteins) that act as catalysts and help complex reactions occur everywhere in life. Let's say you ate a piece of meat. Proteases would go to work and help break down the peptide bonds between the amino acids.
This document discusses enzymes and their structure and function. It begins by describing the history of the discovery of enzymes and defines them as biological catalysts. It then discusses various topics related to enzymes, including their catalytic mechanisms, structural classifications, active sites, cofactors, nomenclature systems like EC numbers, and factors that influence their activity such as temperature, pH, and substrate concentration. Regulatory mechanisms of enzyme activity are also briefly mentioned.
The document provides an overview of key cellular and molecular biology concepts. It discusses the structure and function of eukaryotic cells and their organelles. Specific topics covered include cell membranes, protein structure and function, lipid and carbohydrate biochemistry, DNA and RNA, gene expression, and central metabolic pathways. The roles of enzymes and metabolic regulation are also summarized.
1) The document describes genetic and recombinant DNA techniques for isolating and characterizing genes, including the study of mutations, DNA cloning, construction of cDNA libraries, and screening libraries.
2) Key techniques discussed are the use of dominant and recessive mutations to identify gene function, restriction enzyme cleavage and ligation to clone DNA, transformation of E. coli to replicate recombinant plasmids, and hybridization to screen libraries with probes.
3) The analysis of mutations, complementation, suppressors, and synthetics lethals are also covered as methods to determine gene function and interactions.
1) The document discusses molecular genetic techniques for isolating and characterizing genes, including the study of mutations, DNA cloning, and recombinant DNA methods.
2) Key terms are defined for genetic analysis, such as alleles, mutants, genotypes, and phenotypes. Methods are described for identifying dominant and recessive mutations through genetic crosses and complementation analysis.
3) Techniques like conditional mutations, suppressor mutations, and synthetic lethal analysis are explained for studying essential genes and protein interactions. DNA cloning is introduced, involving restriction enzymes to cut DNA and ligases to join DNA fragments into vectors.
This document provides an overview of Serial Analysis of Gene Expression (SAGE). SAGE allows for the digital analysis of overall gene expression patterns in a sample by producing a snapshot of the mRNA population. It provides a quantitative and comprehensive expression profile. The document outlines the key principles and steps of the SAGE methodology, including isolating mRNA, synthesizing cDNA, ligating linkers, releasing tags, concatenating tags, and sequencing. It also discusses various applications and advances of SAGE, such as LongSAGE, CAGE, and SuperSAGE. SAGE is a powerful tool for studying gene expression, but it has some limitations regarding transcript identification and quantitation bias.
1. A study found that the bacterial infection Citrobacter rodentium releases extracellular proteinases like trypsin and granzyme A during infectious colitis in mice.
2. These proteinases activate proteinase-activated receptor 2 (PAR2) and induce acute inflammation in the colon through G protein activation and calcium mobilization.
3. Inhibiting the proteinase activity or removing PAR2 reduced colon inflammation, demonstrating the important role of PAR2 activation in the host inflammatory response during enteric bacterial infection.
This document provides an overview of plant biotechnology techniques. It discusses how genes can be manipulated by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using transformation methods like Agrobacterium or gene guns. Transformation cassettes containing the gene of interest and selection markers are used. The document explains this process and provides examples like making crops resistant to herbicides or increasing vitamin levels. It also notes there is public controversy around developing and releasing transgenic organisms.
Biotechnology is the application of living organisms or their components to industrial processes. It involves techniques like genetic engineering, cell culture, and monoclonal antibody production. Biotechnology has applications in medicine like producing insulin, agriculture like genetically modified crops, and forensics like DNA fingerprinting. While it offers benefits, biotechnology also raises societal issues around ethics, safety, and public awareness that are actively debated.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Nucleophilic Addition of carbonyl compounds.pptxSSR02
Nucleophilic addition is the most important reaction of carbonyls. Not just aldehydes and ketones, but also carboxylic acid derivatives in general.
Carbonyls undergo addition reactions with a large range of nucleophiles.
Comparing the relative basicity of the nucleophile and the product is extremely helpful in determining how reversible the addition reaction is. Reactions with Grignards and hydrides are irreversible. Reactions with weak bases like halides and carboxylates generally don’t happen.
Electronic effects (inductive effects, electron donation) have a large impact on reactivity.
Large groups adjacent to the carbonyl will slow the rate of reaction.
Neutral nucleophiles can also add to carbonyls, although their additions are generally slower and more reversible. Acid catalysis is sometimes employed to increase the rate of addition.
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
BREEDING METHODS FOR DISEASE RESISTANCE.pptxRASHMI M G
Plant breeding for disease resistance is a strategy to reduce crop losses caused by disease. Plants have an innate immune system that allows them to recognize pathogens and provide resistance. However, breeding for long-lasting resistance often involves combining multiple resistance genes
2. There are several classes of
proteolytic enzymes.
Serine proteases include digestive
enzymes trypsin, chymotrypsin, & elastase.
Different serine proteases differ in substrate specificity.
For example:
Chymotrypsin prefers an aromatic side chain on the
residue whose carbonyl carbon is part of the peptide
bond to be cleaved.
Trypsin prefers a positively charged Lys or Arg
residue at this position.
serine (Ser, S)
3. During catalysis, there is nucleophilic attack of the
hydroxyl O of a serine residue of the protease on the
carbonyl C of the peptide bond that is to be cleaved.
An acyl-enzyme intermediate is transiently formed.
In this diagram a small peptide is shown being cleaved,
while the usual substrate would be a larger polypeptide.
5. During attack of the serine hydroxyl oxygen, a proton is
transferred from the serine hydroxyl to the imidazole
ring of the histidine, as the adjacent aspartate carboxyl is
H-bonded to the histidine.
The active site in each
serine protease includes
a serine residue, a
histidine residue, & an
aspartate residue.
Asp102
His57
Ser195
Catalytic residues in trypsin
PDB 3BTK
6. Aspartate proteases include
the digestive enzyme pepsin
Some proteases found in lysosomes
the kidney enzyme renin
HIV-protease.
Two aspartate residues participate in acid/base catalysis
at the active site.
In the initial reaction, one aspartate accepts a proton
from an active site H2O, which attacks the carbonyl
carbon of the peptide linkage.
Simultaneously, the other aspartate donates a proton to
the oxygen of the peptide carbonyl group.
aspartate (Asp)
7. Zinc proteases (metalloproteases) include:
digestive enzymes carboxypeptidases
matrix metalloproteases (MMPs), secreted by cells
one lysosomal protease.
Some MMPs (e.g., collagenase) are involved in
degradation of extracellular matrix during tissue
remodeling.
Some MMPs have roles in cell signaling relating to
their ability to release cytokines or growth factors
from the cell surface by cleavage of membrane-bound
pre-proteins.
8. During catalysis, the Zn++
promotes nucleophilic attack
on the carbonyl carbon by the oxygen atom of a water
molecule at the active site.
An active site base (Glu in Carboxypeptidase) facilitates
this reaction by extracting H+
from the attacking H2O.
zinc
water oxygen
Carboxypeptidase
PDB 1YME
A zinc-binding motif at
the active site of a
metalloprotease includes
two His residues whose
imidazole side-chains are
ligands to the Zn++
.
Colors in Carboxypeptidase
image at right: Zn, N, O.
9. Cysteine proteases have a
catalytic mechanism that involves
a cysteine sulfhydryl group.
Deprotonation of the cysteine SH by an adjacent
His residue is followed by nucleophilic attack of
the cysteine S on the peptide carbonyl carbon.
A thioester linking the new carboxy-terminus to the
cysteine thiol is an intermediate of the reaction
(comparable to acyl-enzyme intermediate of a serine
protease).
cysteine
10. Cysteine proteases:
Papain is a well-studied plant cysteine protease.
Cathepsins are a large family of lysosomal cysteine
proteases, with varied substrate specificities.
Caspases are cysteine proteases involved in activation &
implementation of apoptosis (programmed cell death).
Caspases get their name from the fact that they cleave
on the carboxyl side of an aspartate residue.
Calpains are Ca++
-activated cysteine proteases that
cleave intracellular proteins involved in cell motility &
adhesion.
They regulate processes such as cell migration and
wound healing.
11. Activation of proteases:
Most proteases are synthesized as larger pre-proteins.
During activation, the pre-protein is cleaved to remove
an inhibitory segment.
In some cases activation involves dissociation of an
inhibitory protein.
Activation may occur after a protease is delivered to a
particular cell compartment or the extracellular milieu.
Caspases involved in initiation of apoptosis are
activated by interaction with large complexes of
scaffolding & activating proteins called apoptosomes.
See diagram of apoptosome in a Univ. London website.
12. Protease Inhibitors:
Most protease inhibitors are proteins with domains
that enter or block a protease active site to prevent
substrate access.
13. IAPs are proteins that block apoptosis by binding to
& inhibiting caspases.
The apoptosis-stimulating protein Smac antagonizes
the effect of IAPs on caspases.
TIMPs are inhibitors of metalloproteases that are
secreted by cells.
A domain of the inhibitor protein interacts with the
catalytic Zn++
.
Cystatins are inhibitors of lysosomal cathepsins.
Some (also called stefins) are found in the cytosol,
and others in the extracellular space.
Cystatins protect cells against cathepsins that may
escape from lysosomes.
14. Serpins use a unique suicide mechanism to inhibit serine
or cysteine proteases.
• A large conformational change in the serpin
accompanies cleavage of its substrate loop.
• This leads to disordering of the protease active site,
preventing completion of the reaction.
• The serpin remains covalently linked to the protease
as an acyl-enzyme intermediate.
• Movie depicting the conformational changes.
(University of Cambridge website)
• Serpins are widely distributed within & outside of cells,
and have diverse roles, including regulation of blood
clotting, fibrin cleavage, & inhibition of apoptosis.
15. plasma membrane may be processed first in an endosomal
compartment and then delivered into the lumen of a
lysosome by fusion of a transport vesicle.
Solute transporters embedded in the lysosomal
membrane catalyze exit of products of lysosomal digestion
(e.g., amino acids, sugars, cholesterol) to the cytosol.
H+
Lysosome
ATP ADP + Pi
Vacuolar ATPase
low
internal
pH
Lumen
contains
hydrolytic
enzymes.
Lysosomes contain
a large variety of
hydrolytic enzymes
that degrade proteins &
other substances taken
in by endocytosis.
Materials taken into a
cell by inward budding
of vesicles from the
16. Lysosomal proteases include many cathepsins (cysteine
proteases), some aspartate proteases & one zinc protease.
Activation of lysosomal proteases by cleavage may be
catalyzed by other lysosomal enzymes or be autocatalytic,
promoted by the internal acidic pH.
H+
Lysosome
ATP ADP + Pi
Vacuolar ATPase
low
internal
pH
Lumen
contains
hydrolytic
enzymes.
Lysosomes have a
low internal pH due to
vacuolar ATPase, a
H+
pump homologous
to mitochondrial F1Fo
ATPase.
All intra-lysosomal
hydrolases exhibit
acidic pH optima.
17. In autophagy, part of the cytoplasm may become
surrounded by two concentric membranes.
Fusion of the outer membrane of this autophagosome
with a lysosomal vesicle results in degradation of
enclosed cytoplasmic structures and macromolecules.
Genetic studies in yeast have identified unique
proteins involved in autophagosome formation.
autophagosome autophagic
vacuole
(lysosome)
One model
for autophagic
vacuole
formation
18. Protein turnover; selective degradation/cleavage
Individual cellular proteins turn over (are degraded and
re-synthesized) at different rates.
E.g., half-lives of selected enzymes of rat liver cells range
from 0.2 to 150 hours.
N-end rule: On average, a protein's half-life correlates
with its N-terminal residue.
Proteins with N-terminal Met, Ser, Ala, Thr, Val, or
Gly have half lives greater than 20 hours.
Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg
have half lives of 3 min or less.
PEST proteins having domains rich in Pro (P), Glu (E), Ser
(S), Thr (T), are more rapidly degraded than other proteins.
19. Most autophagy is not a mechanism for selective
degradation of individual macromolecules.
However, cytosolic proteins that include the sequence
KFERQ may be selectively taken up by lysosomes in a
process called chaperone-mediated autophagy.
This process, which is stimulated under conditions of
nutritional or oxidative stress, involves interaction of
proteins to be degraded with:
• Cytosolic chaperones that unfold the proteins.
• A lysosomal membrane receptor (LAMP-2A) that
may provide a pathway across the membrane.
• Chaperones in the lysosomal lumen that may assist
with translocation across the membrane.
20. Intramembrane-cleaving proteases (I-CLiPs) cleave
regulatory proteins such as transcription factors from
membrane-anchored precursor proteins.
E.g., precursors of SREBP (sterol response element
binding protein) transcription factors are integral proteins
embedded in endoplasmic reticulum membranes.
21. The released SREBP can then translocate to the cell
nucleus to regulate transcription of genes for enzymes
involved, e.g., in cholesterol synthesis.
S2P (site 2 protease, an I-CLiP) is a membrane-
embedded metalloprotease that cleaves an α-helix of
the SREBP precursor within the transmembrane domain.
NC
membrane
cytosol
lumen
S2P cleavage
releasing
SREBP
SCAP-activated
S1P cleavage
Activation of SREBP
involves its translocation
to golgi membranes
where sequential
cleavage by 2 proteases
releases to the cytosol a
domain with transcription
factor activity.
22. Ubiquitin:
Proteins are usually tagged for
selective destruction in
proteolytic complexes called
proteasomes by covalent
attachment of ubiquitin, a small,
compact, highly conserved
protein.ubiquitin PDB 1TBE
However, some proteins may be degraded
by proteasomes without ubiquitination.
An isopeptide bond links the terminal
carboxyl of ubiquitin to the ε-amino
group of a lysine residue of a "condemned"
protein.
lysine
23. The joining of ubiquitin to a condemned protein is
ATP-dependent.
Three enzymes are involved, designated E1, E2 & E3.
Initially the terminal carboxyl group of ubiquitin
is joined in a thioester bond to a cysteine residue on
Ubiquitin-Activating Enzyme (E1). This is the
ATP-dependent step.
The ubiquitin is then transferred to a sulfhydryl
group on a Ubiquitin-Conjugating Enzyme (E2).
24. A Ubiquitin-Protein Ligase (E3) then promotes transfer of
ubiquitin from E2 to the ε-amino group of a Lys residue of a
protein recognized by that E3, forming an isopeptide bond.
There are many distinct Ubiquitin Ligases with differing
substrate specificity.
• One E3 is responsible for the N-end rule.
• Some are specific for particular proteins.
ubiquitin C S
O
Cys E2 H2N Lys protein to be degraded
ubiquitin C
O
HS Cys E2N Lys protein to be degraded
H
+
E3
+
(Ubiquitin-Protein Ligase)
25. H2N COO−
destruction
box
chain of
ubiquitins
Primary structure of a protein
targeted for degradation
More ubiquitins are added to form a chain of ubiquitins.
The terminal carboxyl of each ubiquitin is linked to the
ε-amino group of a lysine residue (Lys29 or Lys48) of
the adjacent ubiquitin.
A chain of 4 or more ubiquitins targets proteins for
degradation in proteasomes. (Attachment of a single
ubiquitin to a protein has other regulatory effects.)
26. H2N COO−
destruction
box
chain of
ubiquitins
Primary structure of a protein
targeted for degradation
Some proteins (e.g., mitotic cyclins involved in cell cycle
regulation) have a destruction box sequence recognized
by a domain of the corresponding Ubiquitin Ligase.
27. Ubiquitin Ligases (E3) mostly consist of two families:
Some Ubiquitin Ligases have a HECT domain
containing a conserved Cys residue that participates in
transfer of activated ubiquitin from E2 to a target
protein.
Some Ubiquitin Ligases contain a RING finger domain
in which Cys & His residues are ligands to 2 Zn++
ions.
A RING (Really Interesting New Gene) finger is not
inherently catalytic. It stabilizes a characteristic globular
domain conformation that serves as a molecular scaffold
for residues that interact with E2.
28. Regulation of ubiquitination:
Some proteins regulate or facilitate ubiquitin conjugation.
Regulation by phosphorylation of some target proteins has
been observed.
E.g., phosphorylation of PEST domains activates
ubiquitination of proteins rich in the PEST amino acids.
Glycosylation of some PEST proteins with GlcNAc has
the opposite effect,
prolonging half-life
of these proteins.
GlcNAc attachment
increases with elevated
extracellular glucose,
suggesting a role as
nutrition sensor.
29. A ubiquitin-like protein called Nedd8 may be attached
to ubiquitin ligases (E3) that have a "cullin" subunit
including a RING finger domain.
De-neddylation (removal of the Nedd8 protein),
catalyzed by a metalloprotease subunit of a complex
called the COP9 signalosome, activates the E3 ligases.
Some disease-causing viruses target host cell proteins
for degradation in the proteasome.
They either activate a host cell Ubiquitin Ligase to
ubiquitinate host proteins, or encode their own
Ubiquitin Ligase.
30. The proteasome core complex, with a 20S sedimentation
coefficient, contains 2 each of 14 different polypeptides.
7 α-type proteins form each of the two α rings, at the
ends of the cylindrical structure.
7 β-type proteins form each of the 2 central β rings.
Proteasomes:
Selective protein
degradation occurs
in the proteasome,
a large protein
complex in the
nucleus & cytosol
of eukaryotic cells.
20 S Proteasome
(yeast) closed state
two views PDB 1JD2
α
β
β
α
31. The 20S proteasome core complex encloses a cavity with
3 compartments joined by narrow passageways.
Protease activities are associated with 3 of the β subunits,
each having different substrate specificity.
20 S Proteasome
(yeast) closed state
two views PDB 1JD2
α
β
β
α
32. 1. One catalytic β-subunit has a chymotrypsin-like
activity with preference for tyrosine or phenylalanine
at the P1 (peptide carbonyl) position.
2. One has a trypsin-like activity with preference for
arginine or lysine at the P1 position.
3. One has a post-glutamyl activity with preference for
glutamate or other acidic residue at the P1 position.
Different variants of the 3 catalytic subunits, with
different substrate specificity, are produced in cells of the
immune system that cleave proteins for antigen display.
33. The proteasome hydrolases constitute a unique family of
threonine proteases. A conserved N-terminal threonine
is involved in catalysis at each active site.
The 3 catalytic β subunits are synthesized as pre-proteins.
They are activated when the N-terminus is cleaved off,
making threonine the N-terminal residue.
Catalytic threonines are exposed at the lumenal surface.
threonine (Thr)
34. Proteasomal degradation of particular proteins is an
essential mechanism by which cellular processes are
regulated, such as cell division, apoptosis, differentiation
and development.
E.g., progression through the cell cycle is controlled in
part through regulated degradation of proteins called
cyclins that activate cyclin-dependent kinases.
35. Several subunits of the proteasome are glycosylated with
GlcNAc when extracellular glucose is high, leading to
decreased intracellular proteolysis.
Conversely, under conditions of low nutrition, decreased
modification by GlcNAc leads to increased proteolysis.
Thus protein degradation is responsive to nutrition via
glycosylation of Ubiquitin Ligase & the proteasome itself.
36. Many inhibitors of proteasome protease activity are
known, some of which are natural products and others
experimentally produced.
E.g., TMCs are naturally occurring proteasome inhibitors.
They bind with high affinity adjacent to active site
threonines within the proteasome core complex.
TMCs have a heterocyclic ring structure derived from
modified amino acids.
Proteasome inhibitors cause cell cycle arrest and
induction of apoptosis (programmed cell death) when
added to rapidly dividing cells.
The potential use of proteasome inhibitors in treating
cancer is being investigated.
37. Proteasome evolution:
Proteasomes are considered very old.
They are in archaebacteria, but not most eubacteria,
although eubacteria have alternative protein-degrading
complexes.
The archaebacterial proteasome has just 2 proteins,
α & β, with 14 copies of each.
The eukaryotic proteasome has evolved 14 distinct
proteins that occupy unique positions within the
proteasome (7 α-type & 7 β-type).
38. The ends of the cylindrical complex are blocked by
N-terminal domains of α subunits that function as a gate.
Interaction with a cap complex causes a conformational
change that opens a passageway into the core complex.
Regulatory cap
complexes:
In crystal
structures of the
proteasome core
alone, there is no
apparent opening
to the outside.
20 S Proteasome
(yeast) closed state
two views PDB 1JD2
α
β
β
α
39. The 19S regulatory cap complex recognizes
multi-ubiquitinated proteins, unfolds them, removes
ubiquitin chains, and provides a passageway for
threading unfolded proteins into the core complex.
The 19S cap is a 20-subunit 700 kDa complex, also
referred to as PA700. When combined with a 20S core
complex, it yields a 26S proteasome.
Only low-resolution structural information, obtained
by electron microscopy, is available for the 19S cap.
Location and roles of some constituent proteins have
been established.
40. The outermost "lid" of the 19S cap is a ring of eight
proteins.
The innermost "base" of the 19S cap includes a ring of
six members of the AAA family of ATPases.
These are chaperones that carry out ATP-dependent
unfolding of proteins prior to their being threaded into
the core complex.
It is typical of AAA ATPases that they assemble into
hexameric rings
Isopeptidases in the 19S cap disassemble ubiquitin
chains. Ubiquitins can then be re-used.
At least one deubiquitylating enzyme is located
between the lid & base regions of the 19S cap.
41. A simpler archaebacterial cap complex called PAN
consists only of a hexameric ring of AAA ATPases,
comparable to the base of the 19S regulatory cap.
PAN, in the presence of ATP, was found to cause opening
of a gate at the end of the 20S proteasome through which
an unfolded protein could enter.
The base of the 19S cap is assumed to do the same,
although high resolution structural evidence is still lacking.
A high resolution structure has been achieved for a complex
of the 20S proteasome with an 11S regulatory cap.
The 11S cap is a heptameric complex of a protein PA28.
42. The 11S cap allows
small, non-ubiquitinated
proteins & peptides to
pass into the core
complex.
This does not require
ATP hydrolysis.
The 11S cap is dome-
shaped, with a wide
opening at each end.
20 S Proteasome
(yeast), with
11S Regulator
(Trypanosome)
two views
PDB 1FNT
Binding of the 11S cap alters conformation of N-terminal
domains of core complex α subunits, opening a gate into
the proteasome core. For images see a website.
43. There have been many
structural studies of
isolated core complex
with 19S or 11S cap.
Formation of mixed
complexes of
proteasome core
sandwiched between
19S & 11S caps has
been shown by EM.
20 S Proteasome
(yeast), with
11S Regulator
(Trypanosome)
two views
PDB 1FNT
In vivo a 19S cap may recognize, de-ubiquitinate, unfold &
feed proteins into a core complex, while an 11S cap at the
other end may provide an exit path for peptide products.
See an animation.
44. Compare with Chime the yeast 20S proteasome core
complex, with and without the 11S regulatory cap.
11S-20S-11S
complex
20 S Proteasome
α
β
β
α