1. Proteomics of Hormone Fragments
by
Roshan Kumar
11/13/2014
in partial fulfilment of
Endocrinology Laboratory, Biology 317
Department of Biology, University of Massachusetts at Boston, Boston, MA 02125`
2. Abstract
Artemin and LTBP beta 1 are both proteins that are expressed along the same amino acid
sequence yet they have different functions.1) A protein hormone was assigned to each individual
in the class for further analyses. (Campbell, Fall 2014) 2) This sequence was then explored for
protease resistant peptides by testing it in one of several published proteolysis programs and any
peptides that were similar or greater than 8 amino acids long that survive serial exposure to
multiple proteases were recorded, identified in the parent sequence, and was used in the next step
of the analysis. The resistant peptides that resulted from sequencing, were then used in the search
sequences in classical protein BLAST searches of all existing proteins in the NCBI protein
databases (Campbell, Fall 2014).The searches came up with proteins that contained a similar
sequence to the ones being tested. The protein LTBP beta 1 was found through this process. The
original amino sequence which was Artemin, focused on the development of neurons in central
and peripheral nervous system while LTBP-1 focused on the linkage between the latent form of
TGF beta-1 and the extracellular matrix to ensure that cell functions in rhythm to its surrounding
while inducing apoptosis in cells and maintaining homeostasis l in cells. Thus even though
LTBPβ-1 came from the same amino acid sequence they both had different roles to play thus
signifying that a protein is capable of carrying out a variety of functions that differ from the
function of the original amino acid sequence which was only revealed after it went through
protease cleaving and then analyzing the residual peptides that could not be broken down to see
that the remaining peptide contained a different form of expression that had a different function
from the first protein.
Introduction:
The purpose of this experiment of this experiment is to analyze and find whether peptide
hormones that have been broken down through a stimulated proteolysis of a protein hormone still
show biological activity differentiates them from the original function of the protein hormone.
Proteomics which was used in this lab to study the protein being expressed is defined as the large
scale study of a specific proteome. It includes information on their variations, protein abundance
and their modifications which are all necessary to study how they work and interact in their
environments. Proteome refers to the entire complement of proteins, including the modifications
made to a particular set of proteins, produced by an organism or a cellular system1.( Cancer.gov).
Proteins when expressed by cells, respond by inducing the movement of these endocytotic
vesicles towards the cell nucleus which causes the lowering of pH levels in the vesicles. And by
inducing a change in the pH levels of the vesicles in the cell, this enables some of the proteases
to have a faster effect on the cell when compared to other proteases with each proteases being
adapted to respond to different pH levels to have the maximum effect in breaking down the
hormones. If the protein hormones are not broken down to very small peptides or single amino
acids, the residual peptides may themselves possess hormonal or protein modulatory
actions.2(lab manual).So to study this phenomenon, we used an amino acid sequence from a
chosen protein hormone to analyze for protease resistant peptides that was then run in a database
1 Cancer.gov
2 Endocrinology lab manual
3. against various proteins. The protease resistant peptide that was gained from this was the latent
TGF-β1 binding protein (LTBP-1). The LTBP-1 is a secreted multi domain glycoprotein which
is a component of the TGFB protein complex. It contains 8 cysteine domains with 17 epidermal
growth factors that help it to bind to the receptors. It’s hinge domain which contains putative
herapin binding sequence which is sensitive to proteolytic cleavage.3 It is assumed that LTBP-1
may be involved in the assembly, secretion and targeting of TGFB1 to sites at which it is then
stored and activated.4 It is also theorized to play critical roles in controlling and directing the
activity of TGFB1 while having a structural role in the extra cellular matrix (ECM). 5(Uniprot).
The analyses of the protein hormone proteolytic peptide that was gained from the
breakdown of the original amino acid sequence should possess hormonal and/or protein
modulatory actions.
Materials and Methods
Uniprot and NCBI databases were the resources that were used in finding the protein
matches for the peptide proteins. Uniprot is a comprehensive, high-quality and freely accessible
database of protein sequence and functional information, many entries being derived from
genome sequencing projects. It is used for references to biological functions of proteins
mentioned in research literature. National Center for Biotechnology Information (NCBI) is a
series of databases that contains information related to biotechnology and biomedical fields. In
this lab, the NCBI was used for running resistant peptide sequences through the protein Blast
feature for similarities to existing proteins.
The lab was organized through the following methods.1) A protein hormone was
assigned to each individual in the class for further analyses. The full length amino acid sequence
of the protein hormone was cut and then pasted to Protein Cyberase software. 2) This sequence
was then run against one of several established proteolysis programs that make it easier to
predict fragments when running mass spectral analyses of proteins.6 Peptides that contained
more than 8 amino acids and which survived the exposure to multiple proteases were then
recorded, identified in the parent sequence, and then used in the next step of the analysis.7 3) The
resistant peptides that remained, were then used in the search sequences in classical protein
BLAST searches of all existing proteins in the NCBI protein databases. After setting the
required parameters to go along with the existing settings already established ion the NCBI
database, the analyses was run through the database for results. The search produced a list of
proteins that possessed linear segments that were either identical to the peptides being queried or
were similar in physicochemical properties as would occur by conservative amino acid
substitutions during protein evolution8. Matches that showed a high similarity to the peptide were
3 Journal of Biological Chemistry
4 Uniprot
5 Uniprot
6 Endocrinology Lab manual
7 Endocrinology Lab Manual
8 Endocrinology Lab manual
4. more likely to take longer than matches that were less likely to happen. The top matches from
these searches were then recorded.
Results:
Sequencing of Artemin which had an amino acid sequence of
SLGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSGRARRPPPQPSRPAPPPPAPPSALP
RGGRAAR through the proteolysis program, resulted in multiple protease resistant peptides.
Out of these, the sequence of PPPQPSRPA which had an amino acid length of 9 while starting at
11 and ending at 19 on the amino acid sequence was chosen and then run through NCBI protein
database to produce a sequence that contained proteins found in humans. From these alignments,
the latent transforming growth factor-binding protein was selected with a expect value of 1.3.
The sequence ID was then run through Uniprot database for matches to current proteins found in
humans and a match was found for the latent-transforming growth factor beta binding protein 1
in humans.
Figure 1
This figure is representation of the original amino acid sequence Artemin that was
presented for sequencing. The component in the middle is the neurotrophic factor called
Artemin. It is a ligand for the GFR-alpha-3-RET receptor complex as shown in the figure above
5. that supports the survival of sensory and sympathetic peripheral neurons in culture and also
supports the survival of dopaminergic neurons of the ventral mid-brain9.
The amino acid sequence that was presented in lab for proteolytic analysis was Artemin.
Artemin is a part of glial cell line derived neurotrophic factor (GDNF). The GDNF ligands are
crucial for the development and maintenance of many neurons. Artemin may also act as a
survival/trophic factor for peripheral neurons either in a paracrine fashion for developing sensory
neurons of the DRG or as a target-derived factor for autonomic innervation of the superior
mesenteric artery10. As noted in figure 1, Artemin is very compatible with GFR alpha 3 but it is
also noted to be able to combine with GFR alpha -1 protein, which also raises the possibility that
although GFRα3–RET is the preferred receptor for Artemin, it may utilize the alternative
GFRα1–RET receptor in vivo, particularly to influence neurons in the CNS (• Baloh, 1998).
Figure 2
This is a diagram of the LTBP and fibrillin structures. This figure shows all the members of the
multi domain glycoprotein family that bind to latent TGF β proteins with each LTBP levels of
affinity for the latent TGF beta molecules shown illustrating the capacity of each LTBP to
regulate latent TGF-β protein.
9 Uniprot Artemin
10 Baloh,Robert H, Malú G Tansey, Patricia ALampe, Timothy J Fahrner, Hideki Enomoto, Kelli S Simburger,
MelanieL Leitner, Toshiyuki Araki,Eugene M Johnson, and Jeffrey Milbrandt."Artemin, a Novel Member of the
GDNF Ligand Family,Supports Peripheral and Central Neurons and Signals through the GFRα3–RET Receptor
Complex." Neuron (1998)
6. Figure 3
In figure 3, the illustration shows the activation of TGFβ signaling taking place due to
release of the TGFβ-1 molecules from the combined degradation of the LTBP-LAP(latency
associated protein) receptor. This illustration shows how LTBP regulates and inhibits the TGF β
pathway until the required signal is received , which then activates the TGF-β to bind to its
receptor thus initiating in the instance, the Smad-dependent and independent signaling. Smad-
dependent signaling regulates fibrogenic target genes such as α-smooth muscle actin (SMA),
collagen, connective tissue growth factor (CTGF), tissue inhibitor of metalloprotease (TIMP-1),
and periostin (Cheng, 2012).
7. Discussion
Artemin belongs to the GDNF ligand family which are known to be crucial in the
development and maintenance the central and peripheral neurons. It is known for preferring the
GDNFα3 receptor but is also capable of binding to GDNFα1 although it is not as effective as it is
with the GDNFα3. Structurally it is disulfide-linked homodimeric neurotrophic factor. . The
Artemin gene is located on chromosome 1p32-33, flanked by markers D1S190-ARTEMIN-
D1S1211(telomeric-centromeric) (• Baloh, 1998). In addition, it was noted that Artemin had the
highest forms of expression among human fetal tissues, kidney and lung thus indicating that
these areas had a high concentrations of peripheral nerves which tended to be where Artemin
was highly expressed. Based on the data gathered from RT-PCR analysis on cDNA samples and
Schwann cell, it was deduced that Artemin is produced by Schwann cells (• Baloh, 1998).
Latent TGF- beta via latent TGF-beta binding protein (LTBP) was identified as playing a
major role in the regulation of extracellular matrix synthesis and degradation as it was noted in
figure 3 where TGFβ was expressed by the extracellular membrane. The latent transforming
growth factor-β proteins are secreted multidomain glycoproteins, three of which are central to
TGF-β regulation (1, 2)(Rifken).It stimulates the synthesis of extracellular matrix components
while also preventing the degradation of the matrix by down regulating the expression of
proteinases. TGF-beta 1 is itself a component of the extracellular matrix and is produced by
proteinase thus acting as negative feedback regulator of the proteinases and a positive feedback
mechanism of the extracellular matrix formation (Taipale, 1993). Models indicate that
transglutaminase which is an enzyme that catalyzes the formation of an isopeptide bond between
the extracellular matrix and the latent TGF-beta-1 and is required for the activation of TGF-beta
1 (Taipale, 1993). The LTBP protein is covalently linked to the extracellular matrix by either
disulfide bonds or reducible/non-reducible cross links formed by matrix cross linking enzymes
(Taipale, 1993). The active form of a TGF-beta 1 protein is a highly hydrophobic and positively
charged protein which cause it to bind to many other proteins with affinity via LTBP (Taipale,
1993). It was noted that even though fibroblasts secreted an excess amount of LTBP only a
fraction of LTBP is associated with TGF beta 1 complex (Taipale, 1993). LTBP is co released
with TGF beta 1 from the extracellular matrix cells by plasmin with the level of LTBP increasing
in correlation with the increased level of plasmin (Taipale, 1993). Based on the comparison of
LTBP released from the cell layer into the supernatant and the amount of total LTBP left in the
cell, indicate that major amount of the LTBP is derived from the extracellular matrix. The partial
inhibition of LTBP binding to the extracellular matrix was due to degradation of other soluble
extracellular matrix that was required for proper matrix assimilation (Taipale, 1993). Thus the
association of latent TGF-beta1 via LTBP to pericellular matrices and their release by
proteinases shows that these proteins participate in areas where concentrated actions of
proteolysis take place such as cell invasion, tissue remodeling and wound healing (Taipale,
1993).
As stated in the hypothesis, even when the main amino acid sequences are broken down
in the protease resistant residual peptides, they still demonstrated a significant level of biological
activity. For example when the protein Artemin was broken down into residual peptides, one of
8. the proteins that resulted from the proteolytic cleaving was a latent TGF beta binding 1
protein(LTBP). LTBPβ-1, as noted in figure 3, LTBP has a different function when compared to
Artemin. LTBP focuses on regulating the Transforming growth factor β protein (TBP) which
controls proliferation, cellular differentiation, smad pathway and various other functions in cells.
LTBP regulates TBP proteins to ensure that rapid proliferation of TBP doesn’t happen since TBP
plays a factor in cell apoptosis and cell proliferation which could lead to cancer. Artemin on the
other hand is a protein which utilizes a receptor system that has a common signaling component
which is used by multiple ligands and also has an additional proteins binds to the ligand for high
affinity and specificity and focuses on the central and peripheral nervous system while LTBP-1
links TBP beta to the extracellular matrix to ensure proper functioning of the cellular system
(Uniprot). Thus even though both proteins exist on the same amino acid, they both display
different functions thus showing different biological activities
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