soccer_5456 wrote:TheWiseGirl wrote:@Phenyl: I just took your practice test, and while it did lean towards the easy side, it was still VERY nicely written. You'll go on to write great tests for Science Olympiad!
I agree!!
I have a question about PARP. After doing research, I became confused over how the apoptosis cascade continues with PARP. What exactly happens? Is it the
overactivation of PARP (after Caspase-3 cleavage at the specific domain) which causes PARP to use all the NAD+ in the cell with PAR systhesis and then ATP depletion in the cell? Or is it the
inactivation of PARP after Caspase-3 cuts the DNA-binding domain from the essential catalytic domain and the auto-modification domain, causing the DNA-binding domain to clog up the SSB?
Aren't these two processes very different? Or do they work in conjunction?
Okay, the reason you became confused is because it is
confusing. I'm working my way through the literature on PARP, and there isn't much of a consensus on how PARP actually comes into play in the apoptosis cascade. It has two established roles: it is cleaved by caspase-3, and it can start its own, separate (caspase-independent) apoptosis pathway by causing mitochondria to produce AIF in response to its PAR synthesis. But it isn't known exactly what the cleavage-by-caspase-3 part does (although let's be clear on this one thing that we do know: caspase-3 cleavage of PARP
inactivates PARP – its purpose is to attach a bunch of poly(ADP-ribose), or PAR, to DNA at sites of single strand breakage, and it can't do that if its catalytic domain, which does the PAR synthesis and attachment part, is no longer capable of getting close to DNA because it's no longer attached to the DNA binding domain).
On the one hand, you have the inactivation of this DNA repair protein, which seems like a logical thing to happen during apoptosis. This rather simplistic explanation of what's going on is the one they went for on the
CBM site, so for the purposes of the event, it's a good place to start.
But on the other hand, studies show that apoptosis still occurs in cells that have been modified to express a mutant PARP that cannot be cleaved by caspase-3 (and in fact, one study found that this
increased the rate of cell death – more on that later), so obviously PARP cleavage is not vital for apoptosis. It's not that the DNA repair pathway that PARP participates in somehow prevents apoptosis from occurring – by the time the cell even gets to the point of caspase-3 cleaving PARP, the cell is already pretty much doomed. Even when PARP continues flagging DNA breaks for repair (which is actually all it does; other proteins do the actual repairing), the cell goes ahead and dies anyway.
One theory is that there's no point in having this protein trying to repair DNA that's doomed anyway, and some later steps of apoptosis require ATP, so PARP (which uses NAD+ in its PAR synthesis) is cleaved to prevent ATP depletion. However, like I said before, cell death occurs even when PARP continues using up the NAD+. The question is (and this question may be answered somewhere in the literature, but I certainly haven't found it yet), do the "later steps of apoptosis" that supposedly require ATP still occur? Or does the cell just undergo lysis without the nice, orderly formation of apoptosomes? Certainly the cell exhibits some usual signs of apoptosis anyway: chromatin condensation, morphological changes, etc. But this ATP-depleted form of apoptosis could be messier and less desirable for the organism in the long run.
Basically it comes down to: caspase-3 cleaves PARP, inactivating its DNA-repair function. This also prevents ATP depletion of the cell. Neither of these things appears to be necessary for apoptosis to occur, but they must somehow be preferable to the alternative.
EASTstroudsburg13 wrote:kDa stands for kilodalton, which is derived from daltons, which are a type of mass unit for atoms. I think they're like atomic mass units, but I'm not super sure.
A Dalton is exactly one atomic mass unit. A kiloDalton is, of course, one thousand Daltons. It's a common unit of measure for protein mass, because essentially all proteins are on the kDa scale rather than the Da scale.
It's also a common way of identifying cleavage products of a protein – for instance, the two subunits of caspase-3 that we've been referring to as chain A and chain B are also known as the p17 and p12 subunits, respectively, because the former is about 17 kDa and the latter is about 12 kDa.
