Protein biogenesis and degradation
MBB443/743
Prof. Michel R. Leroux
Fall semester 2012
Wednesday & Friday 8:30 am – 10:20 am
SECB 1012
Office hours
Office
South Sciences Bldg, SSB6144
Telephone
778-782-6683
e-mail
leroux@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”
Course outline
Wednesdays
& Fridays
- lecture
- student presentation(s)
- no specific tutorial time(s) assigned
- no required text
• 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
Class presentations
assigned at least one week in advance
Guidelines:
1. prepare a 1-page (maximum) summary of the
presentation that is distributed to the class
(please e-mail for distribution)
2. introduce paper, giving enough background
~5 min
information for everyone to understand the paper;
this should include some background information
not found in the paper itself
- also explain the research goals
3. present key experimental data along with brief
explanations of procedures; the data presented can
~15 min
be a subset of all of the paper’s data
4. explain the results obtained
Powerpoint
presentations:
on my computer
If time permits: class discussion
- approaches used were appropriate?
- results were convincing?
- further studies required?
Term paper
based on NSERC grant application
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
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.
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
Exams
There 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 site
www.sfu.ca/~leroux
click on teaching and download lectures and other
stuff (e.g. research proposal info)
lowercase PASSWORD for some files: mbb-sfu
Grading
class no.
date
Exam #1
worth
25%
(or 40%)
14
Oct. 19
term paper
20%
21
Nov. 16
assigned individually
or in pairs
presentation(s)
15%
Exam #2
40%
(or 25%)
26
Total
100%
26
Nov. 30
Central dogma of biology
transcription
DNA
translation
RNA
PROTEIN
cellular
proteins
replication
amino acids,
peptides
folding,
assembly,
targeting
degradation
(turnover) FUNCTIONAL
cellular
proteins
(NATIVE)
PROTEIN
cellular
proteins
regulation of
conformation/
function
Link between biogenesis and
degradation: ‘non-native’ proteins
Non-native (unfolded, misfolded, denatured) protein:
a protein that is not properly folded and is not in a functional state
• 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
both processes can be grouped under the
heading of quality control.
Cellular processes involving
non-native proteins: folding and
assembly
• proteins are synthesized on the
ribosome and must fold/assemble
to become native
folding
- proteins are synthesized as
unfolded polypeptide chains
- folding occurs co-translationally
assembly
- folding (and assembly) to the
native state requires the complete
polypeptide chain
Cellular processes involving
non-native proteins: refolding
cellular stress
Native
protein
heat/cold
proteotoxic
chemicals
intracellular
changes
non-native
(unfolded)
protein
aggregated
protein
various cellular
proteins
Cellular processes involving
non-native proteins: transport
• 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 involving
non-native proteins: regulation of
protein conformation/function
• under certain circumstances, the conformation
(activity) of some proteins must be modulated
inactive,
nonnative
- heat shock transcription factor is activated
only during a cellular stress
cellular
event
- steroid receptors must be kept in a form that
is competent to bind ligand, but is not active
active,
native
- signalling molecules (kinases) are kept in an
inactive conformation until phosphorylated
Cellular processes involving
non-native proteins: degradation
unfolding proteolysis
*
Protein destined
for degradation
proteolysis
*
*
peptides
antigen
presentation
*steps involve various
cellular machineries
amino
acids
Cellular processes involving
non-native proteins: quality control
refolding
non-native
protein
refolding
Native
protein
Native
protein
unfolding
degradation
peptides,
amino
acids
Protein misfolding diseases
diseases
Cystic Fibrosis
Huntington’s disease
Alzheimer’s disease
Parkinson’s disease
Retinitis pigmentosa
cataracts, desminrelated myopathy
cancer
BBS, MKKS
CJD, BSE (‘mad cow’)
sickle cell anaemia
protein(s)
CF transmembrane
regulator (CFTR)
Huntingtin
β-amyloid
α-synuclein
rhodopsin
α-crystallin
p53, VHL
BBS chaperonin
prion protein
haemoglobin
Protein degradation: cellular roles
Degradation involves numerous cellular processes, including:
processing of nascent polypeptides
cleavage of proteins to peptides for antigen presentation
destruction of proteins that are inefficiently folded/processed
degradation of aberrant (mutant) proteins
turnover of cell-cycle or other proteins that are short-lived or
whose presence in the cell is strictly regulated
destruction 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
Protein misfolding disease:
amyloidosis
• at least
16 different
proteins are
implicated in
amyloid
diseases
• a number of
different proteins
can be induced
to form fibrils
in vitro as well
amyloid formation
Amyloid structure
electron microscopy
cross-section
Courtesy of Helen Saibil
Dept. of Crystallography, Birkbeck College London
model of filament
Protein modification
modification
Phosphorylation
Methylation
Hydroxylation
Sulfation
Prenylation
Palmitoylation
Myristoylation
Acetylation
Sulfation
Amidation
Ubiquitination
Truncation
target site
Ser, Thr, Tyr
Arg, Lys, His, Glu
Pro, Lys, Asn, Asp
Tyr
Cys
Cys
N-terminus
Lys, N-terminus
Tyr
C-terminus
Lys
various
cellular process
signalling, activation
prot. repair, chemotaxis
collagen structure
protein-protein intera’n
signalling, oncogenesis
membrane association
membrane association
gene expression
protein-protein intera’n
bioactive peptides
degradation/other
activation
Topics covered
01 introduction: folding/degradation
02 protein structure, translation, etc.
03 protein folding in the cell (I)
04 protein folding in the cell (II)
05 protein folding catalysts
06 paper presentations/discussion
07 chaperones involved in folding (I)
08 chaperones involved in folding (II)
09 the cell stress response
10 protein transport, translocation
11 protein stability, interactions, etc.
12 protein misfolding diseases
13 various
14 exam #1
15 regulation of protein conformation
16 protein modification
17 cellular protein degradation
18 ubiquitin and Ub-like proteins
19 proteasome & other proteases
20 chaperones involved in degrada’n (I)
21 chaperones involved in degrada’n (II)
22 quality control
23 protein degradation diseases
24 various
25 various
26 exam #2