Martin Lab Andy Andreas
<p>May 05, 2015 10:47 PM Andreas = Andy Martin. Prof.</p>
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see his excellent keynote today at 2 PM at Protein QC Conf. in LKS at Stfd.\we chat after.</p>
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MARTIN, Andy, ATP-dependent Protein Metabolism, Cal Berk, May 5 2015
This is at 2 PM in Berg Hall in LKS at Stfd.
A keynote in the Protein quality control conference today.
Hosted by Dept of Chem and Systems Bio at Stfd.
He is a Prof. at Cal, Berk. (many awards)... he begins...</p>
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degenerated proteins (can cause Alz Dis and many others)</p>
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proteolysis is tightly controlled..</p>
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ubiquitin-proteasome system is highly specific.</p>
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he shows a barrel-shaped peptidase... that contains a</p>
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26S proteasome as a compatmental AAA+ protease. which</p>
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depends on compartmental proteases (it does ATP-dependent substrate degradation)</p>
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we use 2 model systems in my lab... he says... one from</p>
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S cerevisiae and also another called ClpXP form E coli... (clip XP)</p>
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start of talk... will focus on E coli ClpXP</p>
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how does mechanical unfolding of the substrate protein work?</p>
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he shows a guy sweating bullets trying to pull a protein thru a small opening... really tough!</p>
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this process is v ATP dependent... unfolding is tough!</p>
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we use a laser trap to study this...</p>
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we monitor bead distances (btwn 2 beads) (similar to Jim Spudich work)</p>
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see report in Maillard in 2011 in Cell.</p>
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we can play tug of war...(to measure the amt of force exerted by the system)</p>
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translocational velocity and stall force of 25 picoNewtons.</p>
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8.5nm/sec is the speed.</p>
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this is the max force that motors can apply..</p>
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<b>it is actively pulling a substrate with power strokes</b>...</p>
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it works in bursts of 2 to 4 nm...</p>
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does the increment of polypeptide (that is pulled thru) dpend on amt of ATP...? (yes)</p>
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he shows a mechanochem model of ClpXP activity...</p>
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the dwell phase of this system is 350 ms. (dwell means its sitting still)</p>
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this is in Nyquist unpublished so far..</p>
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how are subunits coordianted in the hexamer?</p>
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Walker A has a "P-loop" and Walker B...</p>
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this is similar to a 6 cylinder engine...
and what happens when you mess with a cylinder... it decr the rate of work done...</p>
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freq of pauses goes up. (freq of dwell phase)</p>
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if you feed in <b>ATPase dead subunits</b> that causes added pausing...
and decrs the prob of coordianted ATP binding/ hydrolysis.</p>
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each step is 3 nm during ATP binding and hydrolysis....
we next move to a eukarytoic 26S proteasome...</p>
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the most complex protease.</p>
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this is subdivide into 19S regulatory particle and etc.(see youtubes)</p>
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the lid of the proteasome is a 9S subunit for deubiquitinating enzyme DUB.</p>
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and has a 10 subunit base and
a 20S core peptidase.</p>
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we can make these (usu found in eykarytotes) in our lab in E coli...</p>
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see Gabe Lander in Nature in 2012...
revealing the complete subunit architect of the 19S regul particle.</p>
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by Gabe Lander and Eva Nogales.</p>
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step 1 initial substrate binding</p>
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ubiquitin interaction ...</p>
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2) substrate engagement...</p>
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flexible tail (of the polypepetide to be degraded) enters the processing pore</p>
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3) deubiquitationation is tranlocation- dep.</p>
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we do a FRET-based DUB assay...(deubiq assay)</p>
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see his video animations...</p>
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pulls by tail and ubiq comes off and then rest gets pulled in
to degrade it into small peptides.</p>
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see Matyskiela in NSMB in 2013...
for mechs of above.</p>
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looking at conformational. changes upon substr engagement.</p>
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he shows animation of domains movement btwn pre-engagement and engagement of the polypeptide.</p>
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Deubiq is translocation dependent...</p>
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Ubiq in Rpn11 catalytic groove...</p>
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incredible animations/ videos... v detailed. (that are done using free Chimera package from UCSF, he tells me)</p>
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from Worden NSMB in 2014...</p>
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shows Ub cleavage by Rpn11...</p>
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zinc in Ub.</p>
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how can Rpn11 handle thousands of substrates?</p>
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must be able to remove Ub from many many difft poly substrrates.</p>
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uses insert-2 site... to contact K63 (lysine at 63 site)</p>
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Rpn11 is highly promiscuous...</p>
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Rpn11 cleaves only at the substrate and remove entire Ub chain en bloc.</p>
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lid assembly and allosteric regulation of Rpn11...</p>
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DUB is inhibted when lid is isolated... it must be integrated...</p>
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Rpn11 is the key to doing degradation.</p>
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conformational changes during degradation/ engagement.</p>
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The ATPase ring drives conformational changes...</p>
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this is like a spiral staircase...</p>
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substrate engagement by central pore dependes on conformational changes.</p>
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coord conformational changes drive the feeding/ engagement of substrate.</p>
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this is a motor.</p>
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see Sen, Nyquist in Cell in 2013.</p>
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conform changes is a burst that occurs in < 3 ms</p>
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dwell state (rigid state) is abt 350 msec abt 90% of time...</p>
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how is this coord iwth UB removal?</p>
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substrate deubiq controls proteasome dynamics...</p>
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separating allosteric and catalytic effects of Ubp6.</p>
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our goal is to separate Ub processing. from substr engagement and translocation.</p>
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if it cannot pull off Ub then get a stall of proteasome.</p>
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Permanetly Ub-bound Ubp6 abolishes UB-depe degradtion even in a single turnover!!!</p>
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complexity of the proteasome...</p>
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what are functions of extra subunits in the lid?</p>
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goal: characterize substrate processing with and without the lid. of the proteasome.</p>
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there is bimodal switching of lid-base contact btwn engagement and non-engaged state.
may move by 30 angstroms.</p>
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steep spiral staircase of ATP-ase ring.</p>
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model for proteasome substr degradation...</p>
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need to get tail engagement (of substrate)</p>
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ATP ase can tug on it...</p>
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he has a huge lab.... that includes Gabe Lander is at Scripps (as collab)</p>
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Eva Nogales is at UCB.</p>
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Carlos Bustamante is at UCB.</p>
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students: Kris Nyquist
Evan Worden</p>
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must get into AAA ring to be actively pulled on.
force can only be applied after engagement.</p>
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former member: Mary Matyskiela.</p>
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collab Rodrigo Maillard at Georgetown Univ.
(we chat afterward: he does not know whether excess protein in diet may
overwhelm this system (my Q)... another sci thinks autophagy is important
as is mTOR system (eg in life prologation via calorie restriction...
also see Cynthia Kenyon's diet. (eats green veggies; avocados; does eat meat)
Andy says these processes occur intrinsically with intracell products...
(not known whether excess protein leads to excess intracell.)
(but see Ornish remarks abt doubling of Alz disease with incr animal protein in diet).</p>
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