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conflicting results

phosphorylation enzyme

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6 replies to this topic

#1 student47

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Posted 13 September 2014 - 08:12 AM

I have this theoretical question and asked for a explanation of these results. . 

could you help me in answering this.

 

You want to determine the function of a member of an enzyme family capable of phosphorylating other proteins. You do the following experiments:
1) overexpression in fibroblasts – massive substrate phosphorylation – dramatic phenotype
2) knockdown in fibroblasts – substrate phosphorylation unaffected – dramatic, but different phenotype
3) chemical inhibition – substrate phosphorylation unaffected - no phenotype.
These results don’t match. Any idea why



#2 Tabaluga

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Posted 14 September 2014 - 04:01 PM

There is a possible explanation. But what are your own ideas about it ?

As a hint, have you thought about how it can be that substrate phosphorlytion is unaffected despite the inhibition ? Evidently , somehow phosporylation takes place.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 student47

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Posted 14 September 2014 - 11:27 PM

so this was my guess, probably wrong. 

in the first case the more enzyme causes more phosphorylation and makes sense. the second case the phosphorylation is by the other enzymes in the same family. and again in the third case, assuming the chemical inhibition is only for this specific enzyme, this explain the no change in phosphorylation. but the change in phenotype in the second case can be caused by the method of knockdown used. if it is si rna knockdown, it can have off target effects and cause the phenotype. go ahead, please correct me.. 



#4 Tabaluga

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Posted 15 September 2014 - 10:53 AM

Maybe I didn't understand you correctly, but why would the solution we both thought of be wrong ?

The "dramatic, but different phenotype" could be caused by off-target effects, sure, but it could also imply that the protein has other cell functions besides the enzyme function.

Also, after the knockdown this protein is not expressed any more or at least in a reduced amount, while after chemical inhibition the protein is still there, but blocked (we don't know if reversibly or irreversibly, though). If the blocking takes place for example at the catalytic center, this could explain why no enzyme reaction can take place (which is compensated by the other family members) but other cell functions of the protein may be intact, hence "no phenotype" - as opposed to the knockdown which has a "dramatic, but different phenotype" because the other cell functions are also impaired through the lack of protein, which could be indispensable in these functions so as to explain that lack of it leeds to an effect but abundance (as in overexpression) does not show an effect on the other cell functions...

 

 

Just my two cents though....


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#5 student47

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Posted 15 September 2014 - 11:14 AM

i dint mean you are wrong. i meant that i am probably wrong so correct me. i dont have a great deal of understanding in proteomics. i did think of the possibility of other functions, but i did not say it because i would not be able to point to which function since what the phenotype is , is not known. your explanations seem quite plausible, thanks. i just wanted to know what kind of answer is expected of a PhD applicant on an application page in under 250 words, how much in depth analysis or guesswork for missing information etc,  i guess something like what you wrote should have been okey? . 



#6 Tabaluga

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Posted 15 September 2014 - 11:27 AM

i just wanted to know what kind of answer is expected of a PhD applicant on an application page in under 250 words, how much in depth analysis or guesswork for missing information etc,  i guess something like what you wrote should have been okey?

 

I'm afraid I can't estimate that well. Maybe they want to hear a specific solution, but on the other hand if you carefully write your thoughts (or different possible interpretations) and most importantly write   w h y   you came to this interpretation, i.e. which evidence supports it and what might contradict it or support a different hypothesis, you can show them that you can do logical and differential thinking.

Just wanted to caution that I don't guarantee my explanations are the correct ones...bear in mind that especially the last sentence in the above post makes the fairly large assumption that a minimum level of the protein is indispensable for other functions but an abundance of it has no influence.... so if this is really for a PhD application, you might want to go over it with someone else to be on the safe side.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#7 student47

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Posted 15 September 2014 - 11:38 AM

dont worry, the application is already posted and sent yesterday. i just wanted to know the interpretation from someone probably in proteomics field or one knowing of of it better than me probably. if anything your reply has set a kind of idea of logical interpretation in this context. dont bear a guilt over me using this answer, thanks again. 






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