Organization: 12 hours lectures&discussion in class. 2 hours preparation (tutorial) of the personalized experimental Protocols. One day experimental work in the TSABAM of Chemistry (Perlman Building).
Grade: 30% of the grade will be for the active participation, questions, and discussions during the lectures. 10 % of the grade will be for the preparation of the personalized experimental Protocols with the teachers (Dr I. Riven, Dr Y. Barak and myself). 60% of the grade will be based on the quality of the scientific report of the applied experiments performed by the students.
12 hours lectures: Part 1: ~ 6h on the different protein states in health and diseases. Part 2: ~6h on the structure and function of molecular chaperones that control protein states.
Part 1: the different protein states in health and diseases
The problem of protein folding, misfolding and aggregation
Definition of terms: native folding (Anfinsen). Misfolding, aggregation, oligomerization, fibrillation, disaggregation, de-oligomerization, unfolding.
The Native state: The (non-equilibrium) free energy state of native protein structures. Hydrophobic collapse. Why some native protein may be stress labile and others not ?
The Unfolded state: During de novo synthesis, during organelle import, during stress. The transient nature of the unfolded state.
The Misfolded state: An obligate intermediate state on the path of aggregation.
The Aggregated states: Definition, different types of protein aggregates, their free energy state, their reversibility.
The evolution of protein complexity along the tree of life. Why proteins needed to become multi domain, longer and enriched in aggregation-prone beta structures ?
C. Anfinsen showed that native protein folding can be spontaneous.
Possible errors in the spontaneous folding process. Why proteins misfold ? The biochemical characteristics of protein aggregates. Their affinity for other native, unfolded and misfolded proteins and for membranes.
Biological consequences of protein misfolding: loss of biological activity, toxic interactions with membranes, ROS production, apoptosis, tissue loss, aging and neurodegeneration. Examples of alfa-synuclein and PolyQ aggregates
The correlation between cellular toxicity and aggregate size, compactness, solubility, and hydrophobic exposure.
How may some highly soluble IDPs convert into insoluble toxic aggregates enriched in cross beta-structures? Example alfa-synuclein, tau.
The various types of protein aggregates. Soluble, insoluble, protofibrilar, fibrillar, inclusion bodies, liquid phase.
Methods to distinguish aggregates: solubility, Thioflavin-T, FRAP, Lip-mass spec.
How may cells detect the presence of toxic protein aggregates ?
The case of heat sensing: Heat is increasing the propensity of thermolabile proteins to unfold, misfold and form stable aggregates. The membrane thermosensors: TRPV1 in animal and CNGC2/4 in plants
HSPs: proteins that massively accumulate in response to stresses.
NOT ALL HSPs are molecular chaperones and NOT ALL molecular chaperones are heat/stress-induced.
Different types of HSPs:
1) Enzymes that scavenge ROS (APX), 2) metabolic enzymes that produce ROS scavengers (beta-carotene). 3) Enzymes that produce thermo-protecting metabolites (Glycine Betaine, polyols). 4) Holding chaperones (small HSPs that prevent the aggregation of misfolded thermolabile proteins (small HSPs). 5) Unfolding chaperones that revert protein misfolding and thereby disaggregate thermolabile proteins. 6) ATP-fueled proteases that degrade misfolded and aggregated proteins. 7) Transcription factors, such as WRKY and HSFA2 that over produce items 1-6 under stress. A sub-category of HSPs called molecular chaperones, some of which not being heat/stress induced.
Part 2: the structure and function of the main molecular chaperone families
The evolution of HSP20s, HSP60/CCTs, HSP70-JDP, and the HSP70-cochaperones, the NEFs and the HSP70-codisaggregases, ClpB and HSP110 and the HSP70-corefoldase HSP90.
1) HSP20s, AKA small HSPs (structure & function) IBPB, alfa crystalline (structure & presumed functions).
2) HSP60 and GroEL-GroES (structure & function).
HSP60 Mechanism: assist native folding of UNFOLDED polypeptides or unfolds misfolded polypeptides ?
3) Hsp70 and JDPs( and nucleotide exchange factors) (structure & function).
HSP70 Mechanism: ATP-fueled unfolding by clamping and entropic Pulling.
HSP70 as a non-equilibrium nanomachine that can revive native proteins under heat-denaturing conditions.
4) HSP70-codisaggregase ClpB (structure & function).
5) HSP110 (evolution, structure & possible NEF and codisaggregase function).
6) Other Chaperones: TIG, Spy, HSP32
Concluding remarks: the thermodynamics of molecular nanomachines that control protein homeostasis. The role of HSP-chaperones in coping with global warming. Can chaperones avert aging and degenerative diseases? Can chaperones promote the survival of cancer cells ?