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Chaperones involved in protein folding Overview of molecular chaperone families - distribution of chaperones in eukaryotes, archaea and bacteria Nascent-chain binding chaperones - Trigger Factor, NAC, Hsp70, prefoldin
Overview of chaperone families: Distribution - prefoldin prefoldin [Hsp90] - Hsp90 - - Hip, Hop, Bag, clusterin, cofactors A-E, calnexin, calreticulin, etc. etc. [PapD/FimC] - - SecB - - AAA ATPases AAA ATPases AAA ATPases [small Hsps] small Hsps small Hsps chaperonins (Group I) chaperonins (group II) chaperonins (group II) Hsp70 system [Hsp70 system] Hsp70 system - NAC NAC Trigger Factor - - Bacteria Archaea Eukaryotes
Overview of chaperone families: multigene families
not all molecular chaperone families are present in the three domains of life; some are highly specialized and are found in just one domain
eukaryotes have evolved not only more different families of chaperones, but typically have more members ( e.g. , Hsp70, small Hsps, prefoldin, etc.)
related to diversity of processes? (eukaryotes have organelles, greater diversity of cell functions)
must perform comparitive studies, e.g. , with genome of the microsporidian Encephalitozoon cuniculi , 2.9 Mb. Amitochondriate, parasitic; cause of severe infections
bacteria and archaea do have chaperone multigene families
potential overlap in function? ( e.g. , Hsp70 in same/different compartments)
replacement of function by other chaperone families ( e.g. , prefoldin)
COG 15 -------- qv--b-efghs-ujx-l - HslU [ O ] COG1220 ATP-dependent protease, ATPase subunit 48 aomtpkzy--drbc-f-----j ---- SpoVK [ O ] COG0464 ATPases of the AAA+ class 5 58 --- t---yqvdrbcefghsnujxilw ClpA [ O ] COG0542 ATPases with chaperone activity, ATP-binding domain 54 aomtpkzyqvdrbcefghsnujxilw GroEL [ O ] COG0459 Chaperonin GroEL (HSP60 family) 2 26 ------- yqvdrbcefghsnujxilw GroES [ O ] COG0234 Co-chaperonin GroES (HSP10) 6 19 ------- y---rbcefghs-ujx-l - HtpG [ O ] COG0326 Molecular chaperone, HSP90 family 70 - o-tp--yqvdrbcefghsnujxilw DnaJ [ O ] COG0484 Molecular chaperones (contain Zn finger domain) 7 ------------- ce---s-uj ---- CbpA [ O ] COG2214 Molecular chaperones, DnaJ class 36 aomtpkzyqvdrbcefg-s---x --- IbpA [ O ] COG0071 Molecular chaperone (small heat shock protein) 3 10 aomtpk-yq ----------------- GIM5 [ O ] COG1730 Prefoldin, molecular chaperone, beta class 9 aomtpkz ------------------- GIM1 [ O ] COG1370 Prefoldin, molecular chaperone, alpha class category: O Post-translational modification, protein turnover, chaperones archaea yeast bacteria “ C lusters of O rthologous G roups of proteins” Homologues: genes that are related in sequence and function Orthologues: cross-species or cross-domain genes that are related in sequence and function Paralogues: homologous genes that were duplicated in the same organism http://www.ncbi.nlm.nih.gov/COG/xindex.html other categories: translation, transription, cell motility, ion transport, etc. etc.
Different sites of action Location of chaperone is very important: cytosol? membrane? organelle? extracellular? periplasmic?
associated with particular structures?
e.g. , calnexin; must be near polypeptide entry?
must bear sequence tag to target it there
chaperonin required for its own folding
e.g. , clusterin binds large number of extracellular proteins
e.g. , PapD/FimC is required for pilus folding/assembly
Example: - misfolded proteins may end up in aggresomes ( e.g ., CFTR) - aggresomes contain various molecular chaperones, including Hsp70 and Hsp40, as well as proteasome components This can potentially cause problems: - researchers expressed mutant CFTR - they then expressed mutant GFP that is normally broken down - saw GFP fluorescence (green) in the cytosol ( i.e. , it wasn’t degraded) - has implications for proteins that aggregate in cell and cause diseases
- most effective peptidyl prolyl isomerase (PPIase)
- behaves as a conventional molecular chaperone, i.e. , can bind non-native proteins
- ribosome-bound (interacts with RNA in the 50S ribosome subunit, but some of it is cytosolic)
- interacts with large fraction of nascent polypeptides (as determined by cross-linking)
- only occurs bacteria (where it is ubiquitous), although other eukaryal/archaeal proteins have FKBP domains
- deletion is not lethal(!) However, deletion is lethal when knock out bacterial Hsp70, which also binds nascent chains
crystal structure suggests that it forms a ‘pocket’ for chains exiting the ribosome
(recall the ‘crouching Dragon’ structure presented in class)
• how do the chaperone binding site and PPIase cooperate?
• what is the exact nature of the polypeptide binding site?
Nascent-chain binding chaperone: NAC Nascent polypeptide Associated Complex (NAC) - eukaryotic protein consists of alpha and beta subunits; archaea have only beta subunit - as with TF, bound to ribosome - does not contain domain resembling a PPIase Primary function: - prevents inappropriate targeting of nascent polypeptides by SRP - if ER signal sequence is present, SRP binds it, causes translation arrest, and docking occurs; co-translational insertion of protein then takes place, and the sequence is cleaved - if ER sequence is not present, NAC prevents SRP from binding to the nascent chain - evidence suggests it may help targeting to mitochondria
NAC function: example experiment Beatrix et al. (2000) J. Biol. Chem. 275, 37838. Fig. 8. NAC complex, but not the individual subunits, prevent inappropriate interaction of SRP with signal-less chains on ribosomes. High salt-stripped 77aaffLuc RNCs (ribosome nascent chains) obtained by in vitro translation in rabbit reticulocyte lysate, and carrying the photo-cross-linker (TBDA-modified lysine-tRNA), were incubated first with excess SRP, then with the individual NAC subunits, bovine NAC, or recombinant NAC as indicated. Samples were irradiated and analyzed by SDS-PAGE and fluorography. Bovine NAC (lane 6) and the reconstituted recombinant NAC (lane 5) both successfully competed with SRP for interaction with a signal-less chain on the ribosome. But neither alpha-NAC (lane 3) nor beta-NAC (lane 4) alone could prevent SRP from interacting with the signal-less nascent chain on the ribosome. 77aaffLuc is the N-terminal 77 amino acids of firefly luciferase lacking an import signal
If NAC is present at the polypeptide exit tunnel, and generally binds nascent chains (except when it is displaced by SRP), could it act as a molecular chaperone?
Is NAC functionally equivalent to Trigger Factor except for the fact it’s not a prolyl isomerase?
Nascent-chain binding chaperone: Hsp70 Found in nearly all compartments where protein folding takes place: - cytosol of eukaryotes (Hsp70) and bacteria (DnaK) - mitochondria (mt-Hsp70) - chloroplast (cp-Hsp70) - endoplasmic reticulum (BiP) - in yeast and nematodes, there are at least 14 different Hsp70’s One surprising exception: - not found in all archaea; this has been viewed as a paradox - reason is that it has been shown to bind nascent polypeptides: - it can be cross-linked to nascent chains in eukaryotes and bacteria - another reason is that it is important for de novo protein folding
Hsp70 is believed to bind and stabilize nascent polypeptides early in their synthesis--preventing misfolding and aggregation
Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for folding ( e.g ., chaperonins)
Hsp70 is also important during cellular stresses (thermotolerance), and has numerous other functions in the cell apart from assisting de novo protein folding. It often works in collaboration with other chaperones, especially Hsp40
Structure of Hsp70 chaperone ATPase domain (homology with actin, which also binds ATP) Polypeptide binding domain with bound peptide ‘substrate’
Structure of entire molecule (~70 kDa) has not been solved
flexible linkage between ATPase and peptide-binding domains, and different conformations of molecule possible
polypeptide-binding domain consists of beta-sheet scaffold; loops possess hydrophobic residues that contact peptide
domain also has an alpha-helical ‘lid’ that is regulated by the ATPase activity
Substrate specificity of Hsp70 Experiment 1. synthesize 13-mer peptides that overlap by 10 amino acids, based on actual protein sequences (spacer is Ala 2 ) - this covers entire protein sequence and any binding site 2. cross-link peptides to nitrocellulose membrane (automated) 3. add chaperone and allow binding to equilibrium 4. electro-transfer any Hsp70 bound to peptides onto membrane 5. probe membrane by Western blotting with specific antibody 6. screen 37 different proteins this way 7. obtain statistically significant information on binding motif
Hsp40 may bind nascent polypeptides directly, passing these on to Hsp70
although it is a molecular chaperone in its own right, it seems to operate mostly in conjunction with Hsp70
there are numerous Hsp40 homologues in eukaryotes and bacteria; some are specific for the different Hsp70’s, and some actually modulate the function or localization of Hsp70’s
There also exists a number of additional chaperone cofactors that modulate the activity of Hsp70’s:
- e.g. , Hip and Bag; these affect ATPase activity of Hsp70
in yeast, zuotin is an RNA-binding Hsp40 chaperone that is ribosome-bound; a cytosolic Hsp70 interacts with it to bind to nascent polypeptides
Nascent-chain binding chaperone: prefoldin Discovery - a group performed a screen for yeast genes that were synthetically lethal in combination with a gamma-tubulin mutation - found 5 genes that when disrupted, resulted in cytoskeleton defects • actin: sensitivity to osmotic stress, latrunculin-A; disrupted actin filaments • tubulin: sensitivity to benomyl; disrupted microtubules - another lab independently purified a bovine protein complex containing 6 proteins that could bind unfolded actin and tubulin; the yeast complex was later purified and shown to possess the same 6 orthologous proteins as the bovine complex Characterization - synthetic lethality with various actin and tubulin mutants, as well as mutants involved in microtubule processes ( i.e. , cofactors A-E) - may cooperate with cytosolic chaperonin (CCT) in actin and tubulin biogenesis
Prefoldin subunit structure Predicting coiled coils in proteins: - a number of web-based programs are available - rely on the repeating unit of the coiled coil - a and d positions in a-g heptad repeat are usually hydrophobic - the a and d positions form the apolar interface between the two helices; because of alpha helices normally have 3.6 residues/turn, the 3.5 residues/turn of the coiled coil induces a strain on the helix Some coiled coils can have three or more helices
Prefoldin is found in all archaea but Hsp70 is not; those that have Hsp70 probably acquired it via lateral gene transfer
Mechanism of prefoldin is clearly different from that of Hsp70, but the overall function of each may be similar:
- both bind nascent polypeptides
- prefoldin can stabilize an unfolded protein for subsequent folding by chaperonin
(explanation in class)
- range of proteins archaeal prefoldin stabilizes is considerable: 14-62 kDa
Archaeal prefoldin (with 2 different subunits) may play a general role in protein folding whereas the eukaryotic chaperone (with 6 different subunits) may have acquired more specialized functions; this is seemingly the case for the eukaryotic chaperonin CCT, which has 8 different subunits compared to the archaeal chaperonin, which has 1 or 2 subunits, and the bacterial chaperonin (GroEL), which has 1 subunit
the presence of prefoldin may resolve the paradox that many archaea don’t have Hsp70, the otherwise ubiquitous molecular chaperone