This document discusses cysteine proteases and their role in programmed cell death (PCD) in plants. It notes that vacuolar processing enzyme (VPE), a cysteine protease, has been identified as a key executor of PCD in plants, playing a role similar to caspases in animal apoptosis. VPE is involved in plant responses to stresses like radiation and pathogens by degrading vacuolar membranes and releasing hydrolytic enzymes to break down cellular components. The document examines the mechanisms and pathways of PCD in both animals and plants.
3. Radiation. Plant Toxicity.
– Research Proposal: Radiation. Plant toxicity. Inhibitors of radiation plant
toxicity.
– Dmitri Popov
– Full-text Research Proposal · May 2015
– Add resources
– File name: RadiationPlantToxicity1.pptx
DOI: 10.13140/RG.2.1.3321.2402
4. Cysteine Proteases.
– Cysteine proteases, also known as thiol proteases, are enzymes that
degrade proteins. These proteases share a common catalytic mechanism that
involves a nucleophilic cysteine thiol in a catalytic triad or dyad.
– Cysteine proteases are commonly encountered in fruits including
the papaya, pineapple, fig and kiwifruit. The proportion of protease tends to be
higher when the fruit is unripe. In fact, dozens of latices of different
plant families are known to contain cysteine proteases
– Domsalla A, Melzig MF (June 2008). "Occurrence and properties of proteases in
plant latices". Planta Med. 74 (7): 699–711. doi:10.1055/s-2008-
1074530. PMID 18496785
5. Proteases.
– Proteases are classified according to their catalytic site into four major classes:
– serine proteases,
– cysteine proteases,
– aspartic proteases
– metallo-proteases.
8. Serine Proteases.
– Serine proteases (or serine endopeptidases) are enzymes that cleave peptide
bonds in proteins, in which serine serves as the nucleophilic amino acid at the
(enzyme's) active site. They are found ubiquitously in
both eukaryotes and prokaryotes. Serine proteases fall into two broad
categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-
like.
– In humans, they are responsible for co-ordinating various physiological
functions, including digestion, immune response, blood coagulation and
reproduction
9. Bacterial Cysteine Proteases.
– Proteases are enzymes that hydrolyze a peptide bond in proteins and peptides. The
enzymes are essential for the homeostatic control in both eukaryotes and
prokaryotes; however, they produced by pathogenic microorganisms occasionally
act as toxic factors to the host. Although proteases are classified into four groups,
aspartic, cysteine, serine and metallo-proteases, many of the toxic proteases are
metallo-proteases containing a zinc (II) ion in the catalytic center. The progress in
molecular biology has provided much information on the DNA-derived amino acid
sequences for metallo-proteases and has revealed the consensus sequence His-Glu-
X-X-His as the zinc-binding motif. This motif was found in clostridial neurotoxins,
Bacteroides fragilis enterotoxin (Fragilysin) and Bacillus anthracis lethal factor.
– http://www.mdpi.com/journal/toxins/special_issues/toxins-proteases
10. Bacterial cysteine proteases.
– These bacterial toxins show remarkably specific proteolytic actions toward a target
host protein. For instance, clostridial neurotoxins can cleave the protein
components of the neuroexocytosis machinery, which leads to the blockade of
neurotransmitter release and consequent muscle paralysis. Hemorrhagic toxins
from snake venoms are also metallo-protease. A novel cytotoxin from some
enterohemorrhagic Escherichia coli strains consists of one A subunit and five B
subunits. The A subunit is a subtilase-like serine protease. This special issue deals
with various aspects of protein toxins acting as proteases, which include
biochemical and pathological properties, molecular modes of the toxic actions, the
development of inhibitors to prevent or interrupt the toxic actions, and application
to the cell biology.
– http://www.mdpi.com/journal/toxins/special_issues/toxins-proteases
11. Bacterial cysteine proteases.
– Prof. Dr. Shin-ichi Miyoshi
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama
University 1-1-1, Tsushima-Naka, Kita-Ku, Okayama-City, Okayama 700-8530,
Japan
Website: http://www.pharm.okayama-u.ac.jp/en/department/miyoshi/
Fax: +81 86 251 7926
Interests: bacterial protein toxins; pore-forming toxins; cell membrane
proteins/receptors; proteolytic enzymes.
– http://www.mdpi.com/journal/toxins/special_issues/toxins-proteases
12. Cysteine Proteases.
– Proteolysis in plants is a complex process involving many enzymes and
multifarious proteolytic pathways in various cellular compartments, with
cysteine proteinases playing an essential role.
– Małgorzata Grudkowska and Barbara Zagdańska.
13. Cysteine Proteases.
“Cysteine proteinases also referred to as thiol proteases play an essential role in
plant growth and development but also in senescence and programmed cell
death, in accumulation of storage proteins such as in seeds, but also in storage
protein mobilization. Thus, they participate in both anabolic and catabolic
processes. In addition, they are involved in signalling pathways and in the
response to biotic and abiotic stresses. In this review an attempt was undertaken
to illustrate these multiple roles of cysteine proteinases and the mechanisms
underlying their action.”
Małgorzata Grudkowska and Barbara Zagdańska
14. Cysteine Proteases
– However, the activities of cysteine proteinases respond dramatically to different
internal and external stimuli and in some cases they rise to 90% of the total
proteolytic activity
– (Wiśniewski & Zagdańska, 2001).
– Radiation induce proteolytic activity of proteases.
15. Proteolytic enzymes. Venom.
– Venoms.
– Certain types of venom, such as those produced by venomous snakes, can also
cause proteolysis. These venoms are, in fact, complex digestive fluids that begin
their work outside of the body. Proteolytic venoms cause a wide range of toxic
effects, including effects that are:
– cytotoxic (cell-destroying)
– hemotoxic (blood-destroying)
– myotoxic (muscle-destroying)
– hemorrhagic (bleeding) https://en.wikipedia.org/wiki/Proteolysis#Venoms
16. Plant lysosomes?
– Recent evidence suggests that some plant cells contain structures that fit the standard description of lysosomes, both in terms of
what they are and what they do.
– However, the question of the presence or not of lysosomes in plant cells has been discussed by plant biologists for many years*.
There have been increasing reports of plant vacuoles that contain the enzymes found in animal lysosomes, so effectively
'plant lysosomes' being found. Nevertheless ,many textbooks describe lysosomes only in the context of animal cells, e.g. human
epithelial cells, and do not explicitly state whether or not lysosomes are also present in plant cells.
Conventionally, the word "lysosome" is used to refer to vesicular organelles that meet certain criteria and are found in animal cells,
while the word "vacuole" is used to refer to similar organelles that are found in plants, fungi and algae. The controversy centres
around the structural and functional similarity, with experts holding differing views about whether or not the 'similar' organelles in
animals and plants are actually the same, or merely similar.
– The answer to the question 'do plant cells contain lysosomes?' remains controversial.
Conservative statements likely to be acceptable to those of all views about this include:
– Lysosomes are present in animal cells.
– Lysosomes are present in some eukaryotic, but not prokaryotic, cells.
– Some cell biologists state that lysosomes are not present in plant cells. However, in recent years scientists have reported finding
organelles in plant cells that meet the criteria, or most of the criteria, normally used to describe lysosomes in animal cells.
– * E.g. Matile (1968) "Lysosomes of root tip cells in corn seedlines." Planta 79: 181-196
17. Plant vacuoles with serine
proteases.
– DOI: 10.13140/RG.2.1.3185.6400
– A plant vacuolar protease, VPE, mediates radiation-induced hypersensitive cell
death.
– A high level of Plant Vacuolar Protease, activated after moderate and high
doses of radiation, possible playing role as radiation toxin and can induce
development of Acute Radiation Syndromes in mammals after ingestion.
18. Serine Proteases.
– Plant radiation resistance protects plants from radiation in two ways:
mechanisms and by radiation-induced responses of the immune system.
– Relative to a susceptible plant, radiation resistance is the reduction of radiation
damage on or in the plant, while the term radiation tolerance describes plants
that exhibit little disease damage despite substantial radiation levels.
– Plant Radiation Toxins (possible Vacuolar Processing Enzyme) can induce acute
radiation disease of mammals after digeestion.
– Irradiated living plants with active mitosis can be toxic up to 30 days after
irradiation.
19. Serine Proteases.
– “Apoptotic cell death in animals is regulated by cysteine proteinases called caspases.
Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE
deficiency prevents cell death during hypersensitive response and cell death of
limited cell layers at the early stage of embryogenesis. VPE plays an essential role in
the regulation of the lytic system of plants during the processes of defense and
development. VPE is localized in the vacuoles, unlike animal caspases, which are
localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide
strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.”
– Curr Opin Plant Biol. 2005 Aug;8(4):404-8.
– Vacuolar processing enzyme: an executor of plant cell death.
– Hara-Nishimura I1, Hatsugai N, Nakaune S, Kuroyanagi M, Nishimura M.
20. Serine Proteases.
– Programmed cell death (PCD) occurs in animals and plants under various
stresses and during development. Recently, vacuolar processing enzyme (VPE)
was identified as an executioner of plant PCD.
– “A cellular suicide strategy of plants: vacuole-mediated cell death”
– Apoptosis 2006; 11: 905–911 C 2006 Springer Science + Business Media, LLC.
Manufactured in The United States. DOI: 10.1007/s10495-006-6601-1. N.
Hatsugai et al.
21. Serine Proteases.
– Recently, vacuolar processing enzyme (VPE) was identified as an executioner of
plant PCD. VPE is a cysteine protease that cleaves a peptide bond at the C-
terminal side of asparagine and aspartic acid.
– “A cellular suicide strategy of plants: vacuole-mediated cell death”
– Apoptosis 2006; 11: 905–911 C 2006 Springer Science + Business Media, LLC.
Manufactured in The United States. DOI: 10.1007/s10495-006-6601-1. N.
Hatsugai et al.
22. Programmed cells. Apoptosis.
– Programmed cell death (PCD) is an active, genetically controlled
– process leading to selective elimination of unwanted or damaged cells in
eukaryotes. PCD is essential for growth and development of multicellular
organisms as well as for proper response to environment (Gechev et al., 2006;
Lam, 2004).
23. Plant PCD. AP or Ne?
– Plant PCD is associated with a number of developmental processes including
embryo formation, degeneration of the aleurone layer during monocot seed
germination, differentiation of tracheary elements in water-conducting xylem
tissues, formation of root aerenchyma and epidermal trichomes, anther
tapetum degeneration, floral organ abscission, pollen self-incompatibility,
remodeling of some types of leaf shape, and leaf senescence. (Gechev et al.,
2006; Thomas and Franklin-Tong, 2004).
– Programmed Cell Death in Plants: New Insights into Redox Regulation
– and the Role of Hydrogen Peroxide
– Ilya Gadjev,1,* Julie M. Stone,† and Tsanko S. Gechev*
24. PCD
– Programmed cell death (PCD) is a process by which cells in many organisms die.
The basic morphological and biochemical features of PCD are conserved
between the animal and plant kingdoms.
– Cysteine proteases have emerged as key enzymes in the regulation of animal
PCD.
25. PCD.
– The discovery that cell death is a tightly regulated (programmed) process has stirred
a great deal of interest in its mechanisms.
– Studies of animal systems have shown that the execution of programmed cell death
(PCD) or apoptosis is controlled by a multistep signaling pathway (McConkey and
Orrenius, 1994; Stewart, 1994).
– In plants, PCD has been implicated in xylogenesis (Fukuda,
1996; Groover et al., 1997), in some forms of senescence, and in the hypersensitive
response to pathogens and environmental
stresses (Greenberg, 1996; Mittler and Lam,
1996; Lamb and Dixon, 1997).
26. PCD.
– Although a detailed understanding of how plant cells die is still largely
unknown, recent studies have shown that the apoptotic pathways of the animal
and plant kingdoms are morphologically and biochemically similar (Greenberg,
1996; Levine et al., 1996; Wang et al., 1996).
27. Apoptosis.
– Specifically, the morphological hallmarks of apoptosis include cytoplasmic
shrinkage, nuclear condensation, and membrane blebbing (Earnshaw, 1995;
Martins and Earnshaw, 1997); the biochemical events involve calcium influx,
exposure of phosphatidylserine and activation of specific proteases and DNA
fragmentation, first to large 50-kb fragments and then to nucleosomal ladders
(McConkey and Orrenius, 1994; Stewart, 1994; Wang et al., 1996; O’Brien et al.,
1998).
29. Radiation : VPE and PCD.
– VPE processing system mediates a cellular suicide strategy in plants. In animals, dying cells are
packaged into apoptotic bodies
– and then engulfed by phagocytes. In contrast, because plants do not have phagocytes and the cells
are surrounded by rigid cell walls, plant
– cells must degrade their materials by themselves. VPE, which has caspase-1-like activity, is
accumulated after perception of death signals such
– as pathogen infection. VPE is involved in activation of the target proteins to provoke disintegration
of the vacuolar membranes. Consequently,
– the vacuolar hydrolytic enzymes leave the vacuole for the cytosol and degrade cellular components.
Plants have evolved a death strategy that
– is mediated by the VPE processing system, which is not seen in animals. A cellular suicide strategy
of plants: vacuole-mediated cell death.
N. Hatsugai et al. DOI: 10.1007/s10495-006-6601-1
30. VPE and PCD. Radiation.
– The genome of an organism is under constant attack from endogenous and exogenous DNA
damaging factors, such as reactive radicals, radiation, and genotoxins. Therefore, DNA damage
response systems to sense DNA damage, arrest cell cycle, repair DNA lesions, and/or induce
programmed cell death are crucial for maintenance of genomic integrity and survival of the
organism. Genome sequences revealed that, although plants possess many of the DNA
damage response factors that are present in the animal systems, they are missing some of the
important regulators, such as the p53 tumor suppressor. These observations suggest
differences in the DNA damage response mechanisms between plants and animals. In this
review the DNA damage responses in plants and animals are compared and contrasted. In
addition, the function of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific
transcription factor that governs the robust response to DNA damage, is discussed.
Biology 2013, 2, 1338-1356; doi:10.3390/biology2041338
31. Radiation : VPE and PCD.
– Can irradiated plant’s cells used for feeding induce radiation disease of
mammals?
– Yes.
32. VPE and PCD ( apoptosis or
necrosis)
– A careful analysis by FDA of all Army data present (including 31 loose-leaf
notebooks of animal feeding test results) showed significant adverse effects
produced in animals fed irradiated food...
– http://www.mercola.com/article/irradiated/irradiated_research.htm
– In the course of legalizing the irradiation of beef, chicken, pork, fruit,
vegetables, eggs, juice, spices and sprouting seeds -- a process that has spanned
nearly 20 years -- the U.S. Food and Drug Administration has dismissed or
ignored a substantial body of evidence suggesting that irradiated food may not
be safe for human consumption.
– http://www.mercola.com/article/irradiated/irradiated_research.htm
33. Radiation: VPE and PCD.
– What were these adverse effects?
– A decrease of 20.7 percent in surviving weaned rats.
– A 32.3 percent decrease in surviving progeny of dogs.
– Dogs weighing 11.3 percent less than animals on the control diets... Carcinomas
of the pituitary gland, a particularly disturbing finding since this is an extremely
rare type of malignant tumor."
– Food irradiation: An FDA report. FDA Papers, Oct. 1968
34. Radiation: VPE and PCD.
– Fatal Internal Bleeding in Rats (I)
– "A significant number of rats consuming irradiated beef died from internal
hemorrhage within 46 days, the first death of a male rat coming on the 11th
day of feeding. This rat became sluggish on the 8th day of the regimen and
started refusing food. He continued to be morbid during the next two days, did
not eat any food, lost weight and appeared anemic. He was found dead on the
11th day.
– Vitamin K deficiency in rats induced by feeding of irradiated beef.
– Journal of Nutrition, 69:18-21, 1959. (Cosponsored by the Surgeon General of
the US Army)
35. Radiation: VPE and PCD.
– Fatal Internal Bleeding in Rats (II)
– "Hemorrhagic death had occurred in all males fed irradiated diets by day 34...
There is evidence to suggest that inefficient absorption of vitamins, i.e. vitamin
K, from the intestinal tract may contribute to a deficiency state." [Note: Vitamin
K plays a major role in blood clotting.]
– Influence of age, sex, strain of rat and fat soluble vitamins on hemorrhagic
syndromes in rats fed irradiated beef.
– Federation Proceedings, 19:1045-1048, 1960. (Cosponsored by the Surgeon
General of the US Army)
36. Radiation: VPE and PCD.
– Fetal Deaths in Mice
– "Freshly irradiated diets produced elevated levels of early deaths in [mice
fetuses]... The increase in early deaths would suggest that the diet when
irradiated has some mutagenic potential."
– Irradiated laboratory animal diets: Dominant lethal studies in the mouse.
– Mutation Research, 80:333-345, 1981.
– http://www.mercola.com/article/irradiated/irradiated_research.htm
37. Radiation: VPE and PCD.
– Toxic effects of irradiated foods. Nature, 211:302, 1966.
– A Thalidomide Warning (II)
– "Irradiating can bring about chemical transformations in food and food components resulting
in the formation of potential mutagens, particularly hydrogen peroxide and various organic
peroxides.
– It is now realized, especially since the thalidomide episode, that older testing protocols do not
detect the more subtle population hazards such as mutagens and teratogens. In view of the
serious consequences to the human population which could arise from a high level of induced
mutations, it is desirable that protocols for irradiated food should include in vivo tests on
mammals for possible mutagenicity."
– Mutagenicity and cytotoxicity of irradiated foods and food components.
– http://www.mercola.com/article/irradiated/irradiated_research.htm
38. VPE and PCD.
– Bulletin of the World Health Organization, 41:873-904, 1969. (Cosponsored by the
US Atomic Energy Commission and Food and Drug Administration)
– A Host of Problems
– "Numerous studies have been carried out to ascertain whether cytotoxic effects
occur when un irradiated biological test systems are cultured or fed with irradiated
media or food. In such studies, adverse physiological growth retardation and
inhibition, cytological cell division inhibition and chromosome aberrations and
genetical effects have been observed in a wide range of test systems, ranging from
bacteriophages to human cells... The available data suggest that a variety of free
radicals may act as the toxic and mutagenic agents.“
– http://www.mercola.com/article/irradiated/irradiated_research.htm
39. Radiation Effects. Toxicity of
Plants after irradiation.
– Cytotoxic and mutagenic effects of irradiated substrates and food material.
Radiation Botany, 11:253-281, 1971.
– A Cancer Warning
– "An increase in concentration of a mutagen in food by irradiation will increase
the incidence of cancer. It will take four to six decades to demonstrate a
statistically significant increase in cancer due to mutagens introduced into food
by irradiation. When food irradiation is finally prohibited, several decades worth
of people with increased cancer incidence will be in the pipeline.“
– http://www.mercola.com/article/irradiated/irradiated_research.htm
40. Radiation Effects. Toxicity of
Plants after irradiation.
– Growth, reproduction, survival and histopathology of rats fed beef irradiated
with electrons. Food Research, 20:193-214, 1955.
– Chromosomal Damage to Human Cells (I)
– "Irradiated sucrose solutions were extremely toxic to human white blood
cells. Cell divisions were inhibited. Degenerated cell divisions were observed
and the chromosomes were grossly damaged. The DNA was clumped or the
chromosomes appeared shattered or pulverized. In contrast, treatment with un
irradiated sucrose at the same concentration had no apparent effect on the
mitotic rate and the chromosomes were not visibly damaged.“
– http://www.mercola.com/article/irradiated/irradiated_research.htm
41. Radiation Effects. Toxicity of
Plants after irradiation.
– Cytotoxic and radiomimetic activity of irradiated culture medium on human
leukocytes. Current Science, 16:403-404, 1966.
– Toxic Chemical Formed in Food Containing Fat (I)
– "When food containing fat is treated by ionizing radiation, a group of 2-
alkylcyclobutanones [toxic chemicals] is formed. To date, there is no evidence
that the cyclobutanones occur in unirradiated food. In vitro experiments using
rat and human colon cells indicate that 2-dodecylcyclobutanone (2-DCB)... is
clearly cytotoxic and genotoxic.“
– http://www.mercola.com/article/irradiated/irradiated_research.htm
42. Radiation Toxins.
– Radiation Toxins – Effects of Radiation Toxicity, Molecular Mechanisms of
Action, Radiomimetic Properties and Possible Countermeasures for Radiation
Injury.
– http://www.intechopen.com/books/current-topics-in-ionizing-radiation-
research/radiation-toxins-molecular-mechanisms-of-toxicity-and-radiomimetic-
properties-
43. Pharmaceuticals.
– Currently there is no widespread use of cysteine proteases as approved and
effective anthielmintics but research into the subject is a promising field of study.
Plant cysteine proteases isolated from these plants have been found to have
high proteolytic activities that are known to digest nematode cuticles, with very low
toxicity. Successful results have been reported against nematodes such
as Heligmosomoides bakeri, Trichinella spiralis, Nippostrongylus
brasiliensis, Trichuris muris, and Ancylostoma ceylanicum; the
tapeworm Rodentolepis microstoma, and
the porcine acanthocephalan parasite Macracanthorynchus hirundinaceus.
– A useful property of cysteine proteases is the resistance to acid digestion, allowing
possible oral administration. They provide an alternative mechanism of action to
current anthelmintics and the development of resistance is thought to be unlikely
because it would require a complete change of structure of the helminth cuticle.
45. Literature.
– Otto, H.-H. & Schirmeister, T. (1997) Cysteine proteases
– and their inhibitors. Chem. Rev. 97,
– 133–171.
46. Literature.
– Structural studies of cysteine proteases and their inhibitors.
– Acta Biochimica Polonica. Vol. 48 No. 1/2001
– 1–20
– Zbigniew Grzonka, Elibieta Jankowska, Franciszek Kasprzykowski,
– Regina Kasprzykowska, Leszek £ankiewicz, Wies³aw Wiczk, Ewa Wieczerzak,
– Jerzy Ciarkowski, Piotr Drabik, Robert Janowski, Maciej Kozak,
– Mariusz Jaskólski, and Anders Grubb.
47. Literature.
– Multifunctional role of plant cysteine proteinases
– Małgorzata Grudkowska and Barbara Zagdańska
– Acta Biochimica Polonica.
– Vol. 51 No. 3/2004
– 609–624.
48. Literature
– Stepek G, Behnke JM, Buttle DJ, Duce IR (July 2004). "Natural plant cysteine
proteinases as anthelmintics?". Trends Parasitol. 20 (7): 322–
7. doi:10.1016/j.pt.2004.05.003.PMID 15193563.
– Behnke JM, Buttle DJ, Stepek G, Lowe A, Duce IR (2008). "Developing novel
anthelmintics from plant cysteine proteinases". Parasit Vectors 1 (1):
29. doi:10.1186/1756-3305-1-29.PMC 2559997. PMID 18761736
49. Literature
– McGrath, M.E. (1999) The lysosomal cysteine proteases.
– Annu. Rev. Biophys. Biomol. Struct. 28,
– 181–204.
– Barrett AJ. (1986) The classes of proteolytic enzymes. In Plant Proteolytic
Enzymes. Dalling MJ, ed, vol. 1: pp 1–16. CRC
– Press, Boca Raton, Fl.
50. Literature
– Kirschke, H., Barrett, A.J. & Rawlings, N.D. (1995)
– Proteinases 1: Lysosomal cysteine proteinases; in
– Proteine Profile 2 (Sheterline, P., ed.) pp.
– 1587–1643, Oxford University Press.
51. Literature
– del Pozo O, Lam E. (1998) Caspases and programmed cell death in the
hypersensitive response of plants to pathogens. CurrBiol.; 8: 1129–32. MEDLINE
– Hara-Nishimura I, Kinoshita T, Hiraiwa N, Nishimura M. (1998a) Vacuolar
processing enzymes in protein-storage
– vacuoles and lytic vacuoles. J Plant Physiol.; 152: 668–74.