MBB443/743MBB443/743

Prof. Michel R. Leroux

Protein biogenesis and degradationProtein biogenesis and degradation

Fall semester 2012

Wednesday & Friday 8:30 am – 10:20 am

SECB 1012

Office hoursOffice hours

Telephone778-782-6683

OfficeSouth Sciences Bldg, SSB6144

e-mailleroux@sfu.ca

• I will be available after the lectures in class or in my office (Wednesday and Friday)

• if you have questions, please try to see me right after class

• any e-mails to me should have the header “MBB443”

• total # classes scheduled: 26

• class presentations: - students give 1-2 oral presentations (in pairs if too many students)- 20 minutes each- the paper(s) will be assigned

• 1 term paper (in pairs if too many students)

• 2 exams

Wednesdays& Fridays

- lecture- student presentation(s)

Course outlineCourse outline

- no specific tutorial time(s) assigned- no required text

Class presentationsClass presentations

2. introduce paper, giving enough background information for everyone to understand the paper; this should include some background information not found in the paper itself- also explain the research goals

~5 min

If time permits: class discussion - approaches used were appropriate? - results were convincing? - further studies required?

~15 min

3. present key experimental data along with brief explanations of procedures; the data presented can be a subset of all of the paper’s data

4. explain the results obtained

Guidelines:

Powerpoint presentations:

on my computer

1. prepare a 1-page (maximum) summary of the presentation that is distributed to the class(please e-mail for distribution)

assigned at least one week in advance

Term paperTerm paperbased on NSERC grant application

Format of manuscript

• 10 double-spaced pages, including references

• 2-2.5 cm margins all around

• 12 pt Times New Roman font

• references should be numbered and have the following style:Deere, J., McIntosh, A. and Crusher, W. (2000) Studies on the refolding of Ribonuclease A. Nature 38, 345-368.

Content

• topic should be related to course material

• Introduction; requires literature search (~4 double-spaced pages)

• research proposal and references (~6 double-spaced pages)

• research proposal should describe a study that would advance our knowledge of biogenesis and/or degradation {or related topic}

- describe goal, importance, procedures (brief), alternate experiments, interpretation of (anticipated) results, conclusions

Important !

• you must use your own words when writing the paper; in the rare case you need to use someone’s wording, use quotations and reference the paper

Timeline• wait about one month before choosing your topic; report due November 16

ExamsExamsThere are two exams

• anything mentioned in class is fair game:

- lectures

- assigned papers (~1 for each term)

- class presentations (no detailed questions)

- in-class discussions

• answering exam questions may also require reading a small portion of a real scientific publication

• mostly short, written answers (a word, a sentence or a paragraph)

• focus is on understanding, not memorization; I will tell you if there is something you should specifically memorize.

• no final exam scheduled Finished on last day of class (Nov. 30) !

Lectures, info available on my web siteLectures, info available on my web sitewww.sfu.ca/~leroux

lowercase PASSWORD for some files: mbb-sfu

click on teaching and download lectures and other stuff (e.g. research proposal info)

worth class no. date

Exam #125%

14 Oct. 19

term paper 20% 21 Nov. 16

presentation(s) 15%assigned individually

or in pairs

Exam #240%

26 Nov. 30

Total 100% 26

GradingGrading

(or 25%)

(or 40%)

Central dogma of biologyCentral dogma of biology

DNADNA RNARNA PROTEINPROTEINtranslation

replication

transcription

FUNCTIONALFUNCTIONAL(NATIVE)(NATIVE)PROTEINPROTEIN

FUNCTIONALFUNCTIONAL(NATIVE)(NATIVE)PROTEINPROTEIN

folding,assembly,targeting

degradation (turnover)amino acids,

peptides

cellularproteins

cellularproteins

regulation of conformation/

function

cellularproteins

Link between biogenesis and Link between biogenesis and degradation: degradation: ‘‘non-nativenon-native’’ proteins proteins

• biogenesis signifies the ‘birth’ of proteins, or the transition between non-native to native states - biogenesis includes: folding, assembly, transport to and across biological membranes, refolding, chemical or structural modification

• degradation represents the ‘death’ of proteins, or the transition from native to ‘non-native’ states to basic constituents- degradation includes the disposal of damaged (non-native) proteins and the timely, regulated turnover of various cellular proteins

Non-native (unfolded, misfolded, denatured) protein:a protein that is not properly folded and is not in a functional state

both processes can be grouped under the heading of quality control.

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: folding and folding and

assemblyassembly

• proteins are synthesized on the ribosome and must fold/assemble to become native

- proteins are synthesized as unfolded polypeptide chains - folding occurs co-translationally

- folding (and assembly) to the native state requires the complete

polypeptide chain

folding

assembly

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: refoldingrefolding

cellular stress

Nativeprotein

non-native(unfolded)

protein

heat/cold

proteotoxicchemicals

intracellularchanges

aggregatedprotein

various cellularproteins

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: transporttransport

• protein transport to, and across biological membranes

- protein must be maintained in a translocation-competent state- protein must not misfold or aggregate- protein must be directed to proper membrane / cellular compartment

• also: intracellular transport (to specific locations in the cytosol, nucleus, etc. (but this typically involves native proteins)

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: regulation of regulation of

protein conformation/functionprotein conformation/function

• under certain circumstances, the conformation (activity) of some proteins must be modulated

- heat shock transcription factor is activated only during a cellular stress

- steroid receptors must be kept in a form that is competent to bind ligand, but is not active

- signalling molecules (kinases) are kept in an inactive conformation until phosphorylated

cellularevent

active,native

inactive,non-

native

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: degradationdegradation

unfolding proteolysis

Protein destinedfor degradation

peptides

antigenpresentation

proteolysis

aminoacids

* * *

*steps involve varioussteps involve variouscellular machineriescellular machineries

Cellular processes involvingCellular processes involvingnon-native proteins: non-native proteins: quality controlquality control

refolding

non-nativeprotein unfolding

degradationrefolding

Nativeprotein

Nativeprotein

peptides,aminoacids

diseases protein(s)

Cystic FibrosisCF transmembrane regulator (CFTR)

Huntington’s disease Huntingtin

Alzheimer’s disease β-amyloid

Parkinson’s disease α-synuclein

Retinitis pigmentosa rhodopsin

cataracts, desmin-related myopathy

α-crystallin

cancer p53, VHL

BBS, MKKS BBS chaperonin

CJD, BSE (‘mad cow’) prion protein

sickle cell anaemia haemoglobin

Protein misfolding diseasesProtein misfolding diseases

Protein degradation: cellular rolesProtein degradation: cellular roles

processing of nascent polypeptidescleavage of proteins to peptides for antigen presentationdestruction of proteins that are inefficiently folded/processeddegradation of aberrant (mutant) proteinsturnover of cell-cycle or other proteins that are short-lived or

whose presence in the cell is strictly regulateddestruction of proteins damaged due to cellular stresses

(oxygen radicals, elevated/reduced temperatures, etc.)turnover of proteins that have lost activity over time

- there are a large variety of proteases in the cell, and degradation is strictly controlled

Degradation involves numerous cellular processes, including:

Protein misfolding disease: Protein misfolding disease: amyloidosisamyloidosis

amyloid formation

• at least16 differentproteins areimplicated inamyloiddiseases

• a number ofdifferent proteinscan be inducedto form fibrilsin vitro as well

Courtesy of Helen SaibilDept. of Crystallography, Birkbeck College London

electron microscopy

Amyloid structureAmyloid structuremodel of filament

cross-section

Protein modificationProtein modificationmodification target site cellular processPhosphorylation Ser, Thr, Tyr signalling, activationMethylation Arg, Lys, His, Glu prot. repair, chemotaxisHydroxylation Pro, Lys, Asn, Asp collagen structureSulfation Tyr protein-protein intera’nPrenylation Cys signalling, oncogenesisPalmitoylation Cys membrane associationMyristoylation N-terminus membrane associationAcetylation Lys, N-terminus gene expressionSulfation Tyr protein-protein intera’nAmidation C-terminus bioactive peptidesUbiquitination Lys degradation/otherTruncation various activationUbiquitination Lys degradation/other

Topics coveredTopics covered01 introduction: folding/degradation

02 protein structure, translation, etc. 15 regulation of protein conformation

03 protein folding in the cell (I) 16 protein modification

04 protein folding in the cell (II) 17 cellular protein degradation

05 protein folding catalysts 18 ubiquitin and Ub-like proteins

06 paper presentations/discussion 19 proteasome & other proteases

07 chaperones involved in folding (I) 20 chaperones involved in degrada’n (I)

08 chaperones involved in folding (II) 21 chaperones involved in degrada’n (II)

09 the cell stress response 22 quality control

10 protein transport, translocation 23 protein degradation diseases

11 protein stability, interactions, etc. 24 various

12 protein misfolding diseases 25 various

13 various 26 exam #2

14 exam #1