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Molecular Week 6 Notes

by: Kiara Lynch

Molecular Week 6 Notes Bio 413

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Kiara Lynch
La Salle

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These notes cover information covered in Week 6. Topics include protein folding, protein digestion, and notes on the paper "Regulation of Transcriptional Activation Domain Function by Ubiquitin."
Dr. Stefan Samulewicz
Class Notes
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This 8 page Class Notes was uploaded by Kiara Lynch on Friday February 26, 2016. The Class Notes belongs to Bio 413 at La Salle University taught by Dr. Stefan Samulewicz in Spring 2016. Since its upload, it has received 18 views. For similar materials see Molecular in Biology at La Salle University.


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Date Created: 02/26/16
Molecular Week 6 Notes Protein Folding  Creating a functional protein o Protein must fold properly (noncovalent interactions) o Bind to cofactors (noncovalent interactions) o Covalent modification by glycosylation, phosphorylation, acetylation, etc. (post-translational modifications) o Assemble with partner protein chains  May need to bind with other proteins, dimerize/tetramize, etc. to become functional  Molten globules o Pliable, not solidified o Some fold on their own o Contains most of the secondary structure of the final form, although alpha helices are unraveled and 1 of the helices is only partially formed o Improper folding leads to a nonfunctional protein; won’t interact with binding partners  Co-translational protein folding o Growing polypeptide chain  N-terminal domain folds while C-terminal domain is still being synthesized  C-terminal domain folds  folding of protein is completed after release from ribosome  Chain has not achieved final conformation when released from ribosome o Growing polypeptide chain acquires its secondary and tertiary structures as it emerges from a ribosome  Protein folding o Each domain of a newly synthesized protein rapidly attains a molten globule state o Subsequent folding is slower and uses chaperone molecules  Heat shock proteins (hsp)  Unregulated when protein starts to unfold  If there is improper folding, the active sites and side chains of the protein are not in the right position. The hydrophobic regions are also exposed due to improper folding.  Chaperone proteins latch onto hydrophobic regions and refold the protein o Proteases recognize improper folding  Hsp70 molecular chaperones o Hsp binding cycles help protein to fold o Unfolding protein  exposed hydrophobic regions  ATP hydrolysis bends to get hydrophobic regions back to center (“more gentle” approach) o Recognizes small stretches of hydrophobic amino acids on protein surface o Aided by a smaller set of hsp40 proteins o ATP bound Hsp70 molecule grasps target protein and hydrolyzes ATP which leads to conformational changes. o Hsp70 associates more tightly with the protein o Hsp40 dissociates  ATP rebinds  hsp70 dissociates after ADP release  Hsp60 molecular chaperones o Barrel shaped; has opening with a lid (GroES cap) o Creates a microenvironment for the protein  Different from aqueous environment in cytoplasm o Protein acquires ability to fold properly o ½ of the complex operates at a time o Misfolded proteins are captured by hydrophobic interactions along rim of barrel o ATP binds and GroES cap closes creating a microenvironment  Protein stuck in confined space o The diameter of the rim increases- stretches and partly unfolds protein o ATP is hydrolyzed and the correctly folded protein is ejected  Process to monitor protein quality o Protein rescue competes with proteasome destruction o Proteasome- barrel filled with proteases  Protein goes in folded and comes out in pieces  Lid recognizes tags for degredation o 3 options for newly synthesized proteins  Correctly folded without help  Correctly folded with help of a molecular chaperone  Incompletely folded forms digested by proteasome Proteasomes  Proteasome Central 20S cylinder o Active sites of proteases inside o 19S cap (recognition particle) at either end o Incorrectly folded proteins are marked for ubiquitlylation for destruction o Uses ATP hydrolysis to unfold polypeptide and feed through narrow channel into inner chamber of the 20S cylinder for digestion to short peptides  Protein digestion by proteasome o Protein in, amino acids out o Ubiquitin  76 amino acid protein tag  Becomes covalently linked to region of target protein  the degron  Lid of proteasome binds to degron and chew apart the protein  Ubiquitin is recycled o Cap marked with ubiquitin chain  translocates protein into proteasome core where it is digested (process mediated by ATP- dependent proteins)  Lid o Hexomeric protein unfoldase o Uses ATP hydrolysis o ATP dependent unfoldase  Recognition tag on lid  Binds to protein with ubiquitin (ub) recognition tag for unfolding  Irreversible conformational changes pull substrate to core and strains ring structure  Pulls apart and moves further into core or stays and dissociates  Unfolded protein moves through pore by ATP hydrolysis  Tag  ATP hydrolysis  unraveled  fed through  Ubiquitin ligases (Ub-ligases) o Large family of proteins with 3 subunits- E1, E2, E3 o C-terminal initially activated by high energy thioester linkage to Cys on E1 protein o E1- activates ubiquitin  Ubiquitin binds to cysteine side chain on E1  ATP hydrolysis powers this rxn o E2- transfers  Ub conjugating enzymes o E2&E3 come together  Ub ligase complex o E3- recognition degron  Marking of proteins by Ub o Each modification has a special meaning to cell  Differ in the way that ub molecules are linked together o Ex: Lys48 linkage signals degredation while Lys63 signals other processes  Inducing degredation of a special protein o There are constantly proteins that need to be degraded so ligases must always be present  Don’t always want them activated o Activation of a ubiquitin ligase  Phosphorylation by protein kinase  Allosteric transition caused by ligand binding  Allosteric transition caused by protein subunit addition o Activation of a degradation signal (activating a degron)  Phosphorylation by protein kinase  Unmasking by protein dissociation  Shielding blocks degron  dissociation unmasks  Creation of destabilizing N-terminus  Remove and expose degron  Summary of the production of a protein by a eukaryotic cell o In nucleus  Initiation of transcription  Capping, elongation, splicing  Cleavage, polyadenylation, termination o In cytosol  mRNA degredation  Initiation of protein synthesis (translation)  Completion of protein synthesis and protein folding  Protein degredation Regulation of Transcriptional Activation Domain Function by Ubiquitin  Many transcription factors are unstable proteins that are destroyed by ub- mediated proteolysis o Covalent attachment of ub to proteins signals their destruction by proteasome  Degron o Domain that signals ubiquitylation o Overylaps closely with a transcriptional activation domain – TAD  Hypothesis- Ub-proteasome pathway is involved in transcription o Tested by examining role of ub in transcriptional activation by VP16 activation domain  Substrate targeting with ub-ligases o Interact with degron and recruit ub-conjugating enzyme to the substrate protein  Identify ub-ligase that targets VP16 TAD in Saccharomyces cerevisiae o Fused VP16 TAD to LexA- DNA binding protein; expressed in yeast o Fused LexA to TAD-degrons from Myc and from yeast cyclin Cln3 o Used pulse-chase analysis (Fig.1a lanes 1-4)  All three TADs acted as degrons and destabilized LexA protein o Examined stability of LexA-VP16  Stabilized by loss of Met30 (Fig.1a)  Met30- substrate recognition component of SCF Ub-ligase family  Specific to LexA-VP16 because deletion had little effect on the stabilities of other 3 LexA fusion proteins  Examined effect of loss of Met30 on transcriptional activation by VP16 (Fig.2) o Modified GAL1 promotor carrying two LexA binding sites that drives expression of beta galactosidase (+ and – Met30) o Measured ability of each LexA fusion protein to activate reporter gene expression (Fig.2a) o Myc, Cln3, and VP16 TADs activated the reporter gene in the presence of Met30 while VP16 TAD did not activate the reporter gene in the absence of Met30.  Determine if loss of Met30 decreases VP16 activity through indirect mechanism o Does LexA-VP16 has activity in Met30-null cells; able to stimulate DNA replication  VP16 activity is not universally blocked in Met30-null yeast  the role of Met30 in VP16 TAD function is specifically related to transcriptional activation  Examined role of Met30 in transcriptional activation by the VP16 TAD o Met30 mediated ubiquitylation of LexA-VP16 may be essential for transcription activation o Circumvent requirement for Met30 by direct ubiquitylation?? o Fused single nonremovable ub to N terminus of LexA-BP16  Protein stability  Did not completely restore destruction but did rescue transcriptional activation (Fig.3b)  Ub and VP16 are required for transcriptional act. In Met30 null cells because if just Ub fuses to LexA, transcription is not activated  Met30 coactivates VP16 by signaling LexA-VP16 ubiquitylation  Met30 mediated ubiquitylation, not destruction, is required for transcriptional activation  Ub function distinct from proteolysis  19S complex- Ub binding module; essential role in transcriptional elongation o Ub recruits 19S complex to promotors to promote transcription elongation  Met30 does not direct LexA-VP16 destruction; activator destruction by the proteasome is a natural consequence of ubiquitylation  Ub dual role in transcriptional activation and activator destruction o Uses TFs to link their activity to their destruction o Non-ubiquitylated activators are stable and inactive o Ub-ligase interaction with activator  activator ubiquitylation  activates transcription factor and primes it for destruction by proteasome o Many transcription factors may be regulated through this mechanism  Gene constructs- inserted into plasmids (circular DNA that genes can be inserted into) then transfected into cells (by freeze-thaw or put in a chamber to shock) LexA HA (epitope tag) LexA VP16 (TAD) HA LexA Cln3 (TAD) HA LexA Myc (TAD) HA Ub Polyhistidine (Hisn) Met30 GST (epitope tag) Transcription start site  _____|LexA|________________[beta-galactosidase] o Beta-galactosidase is a reporter construct  When substrate is added it catalyzes a reaction to produce a visible blue product  Under the control of LexA  When ubiquitin and transcription factors are bound to LexA and are on, beta-gal. turns the products blue  ________||||_____________ o Elements that LexA (DNA binding protein) bind to o Attach TADs to activate transcription  Experimental TAD- VP16  Control TADs- Cln3 and Myc  Met30- ub ligase had GST epitope tag o GST allows for affinity chromatography and pulldown experiment o Polyhistidine tag on ubiquitin used to bind to Nickel  Used antibodies against HA (hemagglutinin) and GST (epitope tags) so that LexA and 3 TADs can be seen on 1 gel with 1 antibody Figure 1a  Asked if Met30 was a specific ligase for VP16  Pulse-chase analysis showed all 3 TADs acted as degrons and destabilized LexA protein (lns.1-4)  LexA-VP16 was stabilized by loss of Met30  Met30- ub ligase; substrate recognition component Figure 1b  Asked what the relationship between Met30 ub ligase and VP16 o VP16 binds Met30  Met30 associated with LexA-VP16 and not other LexA-fusion proteins (lanes 2&4 vs 6)  Top- proves each protein was alone  Bottom- (pull down) Met30 and GST bands shown  Middle- overlap; shows there is an interaction of Met30 with VP16 Figure 1c  Met30 is needed to signal ubiquitylation in VP16 +Met30 -Met30 ∆ Lacks a TAD (degron) LexA region Not ubiquitylated Myc Ubiquitylated Ubiquitylated LexA&My c VP16 Ubiquitylated Not ubiquitylated LexA&VP Met30 is a Ub-ligase that attaches 16 to VP16  1. Vectors with HA-LexA-VP16 and polyhistidine-ub hybid sequences were transfected into yeast cells  2. Ubiquitylated His-LexA-VP16 proteins were purified with nickel chromatography  3. Purified proteins were electrophoresed and immunoblotted  3 fusion proteins were created- LexA attached to 3 different TADs LexA VP16 (TAD) HA (epitope tag) LexA HA LexA Myc HA Ub Polyhistidine (Hin )  Top part of figure- shows where ub was attached  Bottom part of figure- shows the input of proteins; Western blot to show the proteins are there  Smear- shows different sized proteins  ****What happens when ub binds to VP16?**** o Ub is attached in the presence of Met30 and not in the absence of Met30 Figure 2a  Measured ability of each LexA fusion protein to activate reporter gene expression  LexA-VP16 in Met30-null cells is more stable and accumulates at a two-fold higher levels it could not activate transcription  Met30 and VP16 are required for transcriptional activation  With or without Met30, activation took place in Myc and Cln3  Beta galactidase attached to 3 TADs Figure 2b  Asked why transcription didn’t start  PCR of ChIP analysis; fractionalized proteins run on a gel  Chromatin immunoprecipitation analysis (ChIP)- LexA efficiently interacts with promotor DNA in absence of Met30  no global effect location of LexA-VP16 protein o VP16 TAD retained at least 1 function in Met30-null yeast  Row1- fragments with LexA reporter- bound to promoter  Row2- no bands (no association with LexA)  With or without Met30, promotor still binds Figure 2c  Tests stability of the plasmid in the presence and absence of Met30 o No big change in stability (ability to pass on gene to next generation)  VP16 TAD retained ability to stimulate DNA replication Figure 3a  Repeat of 1a but there is no degredation shown in this figure  This is because ubiquitin did not attach correctly so it did not mark for degredation Figure 3b  Shows that ubiquitin attaches for degredation o Needed Met30 to attach ubiquitin  Shows proteins o Met30+ had a larger molecular weight because it is attached to ubiquitin  Compare VP16 and Ub-VP16 in Met30-null cells


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