12: PARPs, Sigma-factors, protein splicing, RNAi in flies, protein highlighting

It's been a while since I've updated, and I've probably forgotten most of the wealth of information I had learned in the past few weeks. Granted, I gave up science for almost a week while I was in Ireland.

So, for one class, I read quite a few papers/reviews about PARPs, or poly-ADP-ribose polymerases. I'm supposed to write a proposal on the topic, which was very difficult, because there's so much speculation:

These enzymes basically tag their targets (which are sometimes even themselves!) with strings of ADP-ribose, in irregular linear and branched ways. The tagging is thought to disrupt protein-protein interactions, but is often transient, as there's an accompanying PARG, or glycohydrase that gets rid of the modificaiton. The best studied PARP is PARP1, which is somehow involved in DNA-damage response. It's also part of a new kind of programed cell death, I think they called it parthanatos (don't quite remember). I got really interested in PARP4 or vPARP, because it's part of this giant organelle-like structure called a vault.

So vaults are ribonucleoprotein complexes about 3x larger than the ribosome, and present in many types of eukaryotic cells at >10,000 copies. They were only discovered in the late 80's, and are composed of major vault protein (MVP), vPARP, TEP-1 (telomerase-like protein), and vRNA. They form this large canister-like structure, but they don't know what it does - many implicate it in drug resistance, transport of proteins between nucleus and cytoplasm, response to cancer-causing compounds, and formation of the nuclear pore complex. Anyway, vPARP isn't essential for forming the structure, and knockout mice are just a bit more sensitive to oncogenic agents, but I want to get an idea about whether the poly-ADP-ribosylation affects formation of these structures. One group even observed that vPARP can form filament-like structures that recruit MVP, so I want to do some in vitro structural work to figure out what the heck is going on!

Recent topics in Chemical Biology class:
A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila
Dietzl, et al. Nature 2007

This collaboration created a GENOME-WIDE RNAi library for flies...seemed pretty useful if you're into that sort of research. They had a pretty easy system for targetting tissue-specific expression, and although they threw around a large number of percentages and controls, it seemed like it was a pretty good tool.

Development of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split Inteins
Shi, and someone else JACS 2005

So, protein-splicing is pretty cool, and I think Tom Muir (whom I interviewed with at Rockefeller) was one of the big proponents of it. Basically, it works like DNA splicing, except with amino acids - the intein is the removeable element, and it catalyzes it's own removal. This paper showed how to put two different inteins together, and provided a way to do FRET within a single protein.

Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting
Ando et al. Science 2004

I thought this was really NEATO! They engineered this fluorescent protein from some sea creature to be able to be reversibly turned on and off, in a FRAP-type way. They then used this to observe the kinetics of protein transport back and forth across the nuclear membrane. They called the protein "Dronpa - after dron, a ninja term for vanishing, and pa, which stands for photoactivation." Gotta love scientists - I don't think I'll ever forget the name of this protein.

We also talked about evolving RNAs to catalyze enzymatic reactions, we read this pointless paper:

Self-Sustained Replication of an RNA Enzyme
Lincoln, et al. Science 2009

where they engineered an RNA ligase to make copies of itself in solution indefinitely - only one problem - they needed to throw in oligonucleotide substrates, so I thought this was worse than the example we talked about in class, where they engineered an RNA to ligate itself and replicate in vitro at least using reverse transcriptase. These experiments are trying to get closer to supporting the RNA world hypothesis, but I would really dislike working on this type of research.

In Micro200, we talked about toxin-antitoxin systems in bacteria, which are a really cool way to force bacteria to hold on to specific genetic sequences. The way most of them work: there are two genes near each other, one encodes a stable toxin, while the other encodes an unstable antitoxin that typically binds to the toxin and sequesters it. Some of the toxins are pretty nasty, for example, MazF is an endonuclease that cleaves any mRNA with an ACA in it (pretty common!) My homework assignment involves this protein in particular - under certain kinds of stress, MazF will selectively kill certain members of an E. coli population while allowing others to live, through an ROS (reactive oxygen species)-dependent and independent pathway. A recent paper suggests that under stress, MazF alters protein expression to favor expression of smaller proteins that may or may not be involved in ROS-defense. Connected to all of this is a quorum-sensing type molecule called extracellular death factor (EDF), a small molecule that they identified as NNWNN. Where does this come from? Apparently, the middle of a random gene (zwf, G6P-1-DH) that gets cleaved (probably via MazF), gets translated as NNWDN, gets acted upon by asparagine synthetase A (AsnA), which converts D to N. Might've been confusing, but REALLY cool. : )

We've also been talking about sigma factors, which are components that assist RNA polymerase in regulating the expression of specific genes in bacteria. These are a way to cluster genes required for adaptation to a particular environmental condition. There are also anti-sigma factors that bind to sigma factors and stop them (there are also anti-anti-sigma factors, and I think even anti-anti-anti-sigma factors, haha, yay life-MAPKKK!)

I read:
A Third Recognition Element in Bacterial Promoters: DNA Binding by the Alpha-subunit of RNA Polymerase
Ross, et al. Science 1993

and

Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation
Zhou, et al. PNAS 1993

which talk about the binding of RNA polymerase alpha-subunit in binding to UP elements, in the region around -40 to -60 before a gene, and then identify specific residues in catabolite gene activator protein (CAP) that bind to DNA and bend it to influence gene expression at the promoter.

Right now? I'm reading:
Structure and function of a transcriptional network activated by the MAPK Hog1
Capaldi, et al. Nature Genetics 2008

probably about transcriptional activator networks in yeast
and
“Proteotyping”: Population Proteomics of Human Leukocytes Using Top Down Mass Spectrometry
Roth, et al. Analytical Chemistry 2008

which sounds self-explanatory.

Dan Kahne gave a good talk on Friday, however, I've probably forgotten most of the details already - he talked about research in his lab on the folding and incorporation into the outer membrane of beta-barrel proteins in gram-negative bacteria. If you think about how these proteins have to first cross the inner membrane and the peptidoglycan layer before getting folded and into the outer membrane, it seems like a pretty difficult process. I've got a few papers lying around that talk about the response bacteria have for detecting misfolding of these proteins, involving DegP or DegS or something, and some more anti-sigma factors that I'll probably learn about in the next day or three. Anyway, there's apparently this complex of 5 proteins, two of which are essential, that are involved in this process. YaeT is one of them, a beta-barrel itself. People still debate about how beta-barrel proteins get into the membrane, because unlike alpha-helical membrane proteins which can probably screw in, beta-barrels will end up having unsatisfied hydrogen bonds at the ending beta sheets before the barrel is closed. Beta-strand augmentation is one strategy Dan thinks the system employs, where the protein attaches a beta strand to YaeT or something. He went through a bunch of data invovling deletion mutants of various periplasmic domains for YaeT, with interesting results. Definitely a problem I want to stay informed about.

I'm learning so much, and it's a shame, because I'm forgetting most of it. I hope all this summarizing on here will help me. : (