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Mateyman Mateyman
wrote...
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Posts: 142
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4 years ago
Hey all!

I am reading this paper about cadherin like protein and how to allows parasite t.vaginalis (STD parasite) to adhere to host cell and ultimately kill the host cell. Here is the link to the article: https://mbio.asm.org/content/10/3/e00720-19

To quickly summarize the article for you guys: they found a cadherin like protein (CLP) on surface of the T.vaginalis parasite and called it TVAG_393390. They determined this protein to be similar to cadherin like proteins cos it had calcium binding sites, also it was structuarlly similar to cadherin proteins. They compared the CLP's binding dependency to calcium (Ca) and found that wild type CLP binds to Ca with high affinity where mutant CLP (CLP-mut) does not bind to Ca with high affinity. Also, they found that when a parasite had a CLP it actually increased adherence to host cell by 3.5 fold compared to parasite without CLP which further proves CLP is similar to cadherin proteins. Also they overexpressed CLP-mut (again remember it doesn't have calcium binding site) they found CLP-mut parasites did not clump together which proves that parasites clump due to calcium binding domains and not cos of the CLP itself.  Overall, parasites with wild type CLP actually kill host cells in 2.35 fold compared to parasites with CLP-mut.

Now that I kinda summarized the paper (feel free to look over abstract) I was given 7 questions to answer after reading the paper, I answered 5 of them and would love if someone double checks my answers, and left the last 2 unanswered and would love some help on that!

1. What type of analysis led to the prediction that the hypothetical protein TVAG_393390 may function as a protein with cadherin-like functions?

I just put that Phyre2 showed that it was similar to cadherin proteins cos it compared that TVAG protein structure to known cadherin proteins and found high match in terms of sequence. But I think main answer is Phyre2, I thought it was BLAST but when I took another look it doesn't seem so, does this answer sound OK?

2.   List at least 3 predicted protein features of TVAG_393390 that supported the prediction that TVAG_393390 may function as a cadherin-like protein (CLP). 

I just put it had 5 extracellular domains, it had calcium binding sites, it was structurally similar. I think this is okay yeah?

3.What did the predicted topology of CLP and the cellular localization of the GFP-CLP reveal? Why would this result be necessary to function as a cadherin-like protein?

See this one I was kinda confused about, if you guys look at figure 2C, it shows the topology, I am not sure what this means though? Like it looks like a trans membrane with N and C terminus and intra and extracellular domains, and the blue things is transmembrane proteins so I get that part. But I don't get why that transmembrane topology is needed in order for CLP to function as a CLP? What does the transmembrane of cadherin proteins actually do? I just put the topology is important cos it shows that the predicted CLP does actually have a transmembrane which further proves its related to cadherin proteins but also transmembrane is important in  cadherin proteins in order to perform cell-cell adhesion. Sounds okay? Also in regards to the localization of GFP-CLP I just put that it showed CLP Is localized on surface of parasites.

4.   What did the adhesion experiments in Fig. 5 and Fig. 6 reveal about CLP’s function? Utilize the terms heterophilic and homophilic interactions in your explanation.

Figure 5 I put that CLP increases host cell binding due to the calcium domain. But I am not sure how to include heterophilic in this figure, would heterophilic apply to CLP-mut?  (They define heterophilic as "interactions involve cadherin proteins binding to a different type of protein on a different cell type") so would CLP-mut be heterophilic cos it has CLP but it doesn't have Ca domain or would the recombinant CLP be heterophilic? I know heterophilic interaction is this figure I  just don't know how.

Figure 6, pretty simple, more CLP = more parasite clumping! While more CLP-mut = less parasite clumping! I can use homophilic interactions here cos those interactions found in cadherin is what is causing the parasites to clump together. I feel like Figure 6 my answer is okay just not sure how heterophillic interaction comes into figure 5.

5.   What molecular approach was used to investigate the role of Ca2+ binding for CLP’s function?   

I believe they used Phyre2 and SuSPect analysis to find the sensitive asparate residues to mutate them.

Then they used isothermal titration calorimeter which measures heat output where they added calcium chloride to wild type CLP and found strong heat changes while CLP-mut and calcium chloride found no heat changes. However its important to note that the CLP wild type produced less heat changes as calcium domain became saturated with calcium chloride so this proves that wild type CLP has only one main domain while other domains do not have affinity, while CLP-mut has no calcium domain cos it didn't produce heat changes.

Anyways they found that calcium affects CLP function in regards to how effective it is to binding to host and to parasite clumping.

I have 2 more questions, but lets cover these first 5, please don't feel rush to help me out or be turned off by the paper, I have to submit this in 1 month from now so I'm chilling so please take your time! I just mainly need help for, Q3 in regards to why topology and cellular localization of the GFP-CLP be necessary to function as cadherin-like protein and for Q4 figure 5. Also maybe Q5 did I get the molecular approach correct? Was it ITC, Phyre2, SuSPect or just ITC? Thanks yall!
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6 Replies
Replies
wrote...
#1
Educator
4 years ago
Quote
2.   List at least 3 predicted protein features of TVAG_393390 that supported the prediction that TVAG_393390 may function as a cadherin-like protein (CLP).

I just put it had 5 extracellular domains, it had calcium binding sites, it was structurally similar

Elaborate on how it's structurally similar.

Quote
3.What did the predicted topology of CLP and the cellular localization of the GFP-CLP reveal? Why would this result be necessary to function as a cadherin-like protein?

See this one I was kinda confused about, if you guys look at figure 2C, it shows the topology, I am not sure what this means though? Like it looks like a trans membrane with N and C terminus and intra and extracellular domains, and the blue things is transmembrane proteins so I get that part. But I don't get why that transmembrane topology is needed in order for CLP to function as a CLP? What does the transmembrane of cadherin proteins actually do? I just put the topology is important cos it shows that the predicted CLP does actually have a transmembrane which further proves its related to cadherin proteins but also transmembrane is important in  cadherin proteins in order to perform cell-cell adhesion. Sounds okay? Also in regards to the localization of GFP-CLP I just put that it showed CLP Is localized on surface of parasites.

Topology means the overall shape, and that really matters if we're dealing with a receptor protein.

Quote
4.   What did the adhesion experiments in Fig. 5 and Fig. 6 reveal about CLP’s function? Utilize the terms heterophilic and homophilic interactions in your explanation.

Figure 5 I put that CLP increases host cell binding due to the calcium domain. But I am not sure how to include heterophilic in this figure, would heterophilic apply to CLP-mut?  (They define heterophilic as "interactions involve cadherin proteins binding to a different type of protein on a different cell type") so would CLP-mut be heterophilic cos it has CLP but it doesn't have Ca domain or would the recombinant CLP be heterophilic? I know heterophilic interaction is this figure I  just don't know how.

Heterophilic means different receptors bind.

Some cell adhesion molecules are homophillic in nature and some are heterophillic. Homophilic means both cells have the same receptors. Heterophilic means different receptors bind. The third way is through linker molecules, fibronectin and lamin, which act as an intermeditary between two receptors which are unable to bind without the link.

Quote
5.   What molecular approach was used to investigate the role of Ca2+ binding for CLP’s function?   

For this, look at the material and methods section, summary the approach they took...

Bring on the rest
Mateyman Author
wrote...
#2
4 years ago
Quote
Elaborate on how it's structurally similar.

I believe CLP has similar tertiary structure to cadherin proteins, thoughts?

Quote
Topology means the overall shape, and that really matters if we're dealing with a receptor protein.

Okay so the question says What did the predicted topology of CLP and the cellular localization of the GFP-CLP reveal? And you said topology is important with a receptor protein which is what CLP is, but to tie that to the question, the predicted topology and the GFP-CLP revealed that the intracellular signaling by CLP is done on the C-terminal tail which is what cadherins C-terminal tail does. Then it asks me why this topology and localization of GFP-CLP is important and I put that this is important to function as a cadherin-like protein because signal transduction events done via the C-terminal of cadherins is what keeps cells in touch and is essential  behind the cell-cell adhesion property of cadherin. Thoughts? You probably have to look at Figure 2 so we are on the same page.


Quote
Heterophilic means different receptors bind.


Some cell adhesion molecules are homophillic in nature and some are heterophillic. Homophilic means both cells have the same receptors. Heterophilic means different receptors bind. The third way is through linker molecules, fibronectin and lamin, which act as an intermeditary between two receptors which are unable to bind without the link.

Yeah I got the definitions, even the papers defines these two terms, but it asks me to apply these two terms to figures 5 and 6. I applied homophillic to figure 6 (look my previous reply) but for figure 5 I don't see how overexpression of CLP increases parasite attachment to host compared to overexpression of CLP-mut which doesn't increase attachment of parasite to host had anything to do with heterophillic...

For final two questions its as follows:

6.Based on the findings of the paper, what would you like to investigate next?  Describe what method/s you would carry out in the laboratory in order to investigate the biological process that you are interested in researching. 

Actually have no clue about this one and I read the paper twice already lol

7. If molecular techniques for T. vaginalis are not limited, what is a loss of function experiment you would propose to further investigate your research question of interest stated in Q#6?  What readout/result would you assess and what would this experiment help you discover?

This one ties back to question 6, again I got no clue, you prob gotta read the paper a little to help me answer these to be honest, would love if you can!
wrote...
#3
Educator
4 years ago
Hi Matey, sorry about the late response, been busy with classes...

Quote from: Mateyman (4 years ago)
CLP has similar tertiary structure to cadherin proteins

That should be stated in the article. If it is, then that's something to add.

Quote
Then it asks me why this topology and localization of GFP-CLP is important and I put that this is important to function as a cadherin-like protein because signal transduction events done via the C-terminal of cadherins is what keeps cells in touch and is essential  behind the cell-cell adhesion property of cadherin.

I like it!

Quote
but for figure 5 I don't see how overexpression of CLP increases parasite attachment to host compared to overexpression of CLP-mut which doesn't increase attachment of parasite to host had anything to do with heterophillic...

Essentially that, one has the receptor, the other doesn't. That's what makes them "heterophilic".

Quote
6.Based on the findings of the paper, what would you like to investigate next?  Describe what method/s you would carry out in the laboratory in order to investigate the biological process that you are interested in researching.

Actually have no clue about this one and I read the paper twice already lol

You need to expand on the study. What was found in the study, and what can you now do with this information moving-forward.

T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis.

From the abstract, you can then compare why CLP is functionally similar to mammalian cadherins. Has that been studied before? If not, we can see what makes them similar, and if there's an evolutionary link between them, and when they diverged.

We can also try to learn what membrane proteins the host expresses that allows this interaction to take place. This ties into question 7, where we can turn-off certain properties of these host membrane proteins to understand how they adhere.

Does that make more sense?
Mateyman Author
wrote...
#4
4 years ago
Makes perfect sense man thank you! And please feel no pressure on anwering these questions I post them a few days before due date just to double check everything before submitting so take your time! The fact that you read these papers is a big thing for me thanks!

Now in regards to Q6: Based on the findings of the paper, what would you like to investigate next?  Describe what method/s you would carry out in the laboratory in order to investigate the biological process that you are interested in researching.

You said
Quote
From the abstract, you can then compare why CLP is functionally similar to mammalian cadherins. Has that been studied before? If not, we can see what makes them similar, and if there's an evolutionary link between them, and when they diverged.

I like you mentioned studying the evolutionary link between CLP and mammalian cadherins but I have no clue how I would go about doing this? Any suggestions? I think building a phylogeny tree using the sequence of CLP to that of mammalian cadherins using something like maximum parsimony which is a bioinformatics technique I just learned about this semester, but I'm not sure it applies here. Any other suggestions for building a phylogeny tree to look at a common ancestor between CLP and mammalian cadherin proteins?

Now Q7 says: If molecular techniques for T. vaginalis are not limited, what is a loss of function experiment you would propose to further investigate your research question of interest stated in Q#6?  What readout/result would you assess and what would this experiment help you discover?

You said
Quote
We can also try to learn what membrane proteins the host expresses that allows this interaction to take place. This ties into question 7, where we can turn-off certain properties of these host membrane proteins to understand how they adhere.

I am not sure how finding the evolutionary link between cadherin link protein shown in the paper and mammalian protein ties this question of performing a loss of function experiment to find out what membrane proteins found in the host

Also to follow up on the loss of function experiment in regards to assessing the result of the experiment, I assume once we turn off some properties off the cadherin like protein and notice that it doesn't adhere to the host anymore it would tell us that that specific property is the main reason behind the adhesion property the CLP has, thoughts? I may have put a confusing question here lol, but I'm just trying to see how to answer the part of Q7 that says "what readoutresult would you assess and what would this experiment help you discover" in regards to performing loss of function on the properties of CLP binded to host cell.
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bio_manbio_man
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#5
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Mateyman Author
wrote...
#6
4 years ago
Thank you!
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