Monday, July 16, 2018

HTRA1 -- The most important protein you never heard of?

Have you ever heard of this HTRA1?!?  If not -- this paper in press at JPR suggests that you (and I) should review the (scant) background literature on it -- or just read this great new study!

What is it? be certain I'm not sure we knew until now. Seems like the literature goes back a few years showing it's important -- but this study shows it's critical -- and what it's doing in our cells!

What's it linked to?
Little things like
-Macular degeneration

You know, nothing serious...but try Google Scholar digging through and wondering how you never heard of something this important. There is SO little. And this group decided to fix it with --- FLOW CELL SORTED QUANTITATIVE PROTEOMICS!! (Yeah!)

EDIT (7/19/18) -- After spending more time understanding the paper. Flow cytometry is being used to verify the cells are in the correct states in their cell cycle. I don't believe active sorting of populations is in play.

They synchronized 2 cancer cell lines, including a modified version of SW480 where they could finely control the amount of HTRA1 protein expression (didn't check details, but published elsewhere). This is where the model gets cooler. They block the cell cycle (with thymidine) using a really interesting 2 stage blocking technique I haven't seen before.

It looks like they block the cells at one stage (the synchronized cells can't continue about their usual business of dividing. They just stop all that stuff.) Then they wash it out in some cells and block the cells again at the next stage in the cell cycle. Sound hard? Sound like the matrix is getting pretty big? You're right on both counts!!

This great study doesn't stop there. They use another checkpoint inhibitor or two. What they get is a big picture of what different levels of HTRA protein does at different points in normal cell cycle stuff. Because the cells are syncrhonized and stopped all together, they have a big population of cells all doing the same thing. Messing with any protein in this system that is linked to cell cycle progression or maintenance is gonna have effects somewhere!

Wait. What? They also throw in some DNA intercalators? This study is big. Wow. Next time someone has any kind of a serious fundamental question about DNA damage or cell cycle checkpoints pull up this paper and --- boom!

(No joke. Go talk to someone who does Radiation Oncology. They'll be really impressed!)

Once this large matrix of cells at all these checkpoint stops were acquired, proteins were extracted and digested and ran on really really long nanoLC columns? I'll need to check on details. I just talked to someone today with a 200cm nLC column I might try, so maybe 380cm isn't actually that crazy. And all spectra were obtained on an Orbitrap Elite. All processing was MaxQuant LFQ and Perseus.

What do you get out of all this work? What about proof that this protein I never heard of appears to be linked to regulating as much as 1/10 of our proteome? And if you adjust this by a very crude estimate of the proteome coverage vs theoretical proteome -- what if it looks almost half as important as the cell cycle indicators that scream "STOP DIVIDING SOMETHING BAD IS HAPPENING TO OUR DNA". For real.

Elegant experimental design -- really clear output -- mysterious LC conditions? Great paper you should check out!

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