Mechanisms of Protein Disaggregation and Turnover by AAA+ Chaperones

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Principal Investigator: Daniel Ryland Southworth
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $402,556
Funding agency: National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT
Protein disaggregation and turnover are essential for protein homeostasis (proteostasis) and cell viability.
Malfunction occurs during cell stress and aging, accelerating deleterious protein aggregation and amyloid
formation. Improved mechanistic understanding is critical for determining how proteostasis pathways fail and for
identifying therapeutic targets in preventing neurodegenerative disease and other protein mis-folding diseases.
Heat shock protein (Hsp) 100 members of the conserved AAA+ family serve critical functions in all life as protein
unfoldases and disaggregases. They form hexameric, ATP-driven machines that catalyze the translocation of
polypeptide substrates through a central channel. The unfolded proteins are then refolded by Hsp molecular
chaperones or degraded by an associated protease, such as in the case of the proteasome.
 Challenges in achieving structures of functional states have led to conflicting mechanistic models across
the AAA+ superfamily. Focusing on conserved Hsp100 members, yeast Hsp104 and the bacterial Clp proteins,
we have overcome these challenges by using cryo-electron microscopy to determine structures of biochemically
defined, functional complexes. We determined the first substrate-bound structures of a AAA+ disaggregase
(Hsp104) in distinct translocation states and discovered these machines operate by a rotary mechanism involving
precise substrate gripping and release states and a two amino acid translocation step. Since our last submission
of this application, we have determined multiple structures of the ClpAP AAA+ protease undergoing active
substrate unfolding and proteolysis Together our discoveries reveal a new paradigm for how AAA+s
mechanically unfold substrates. The next major question to address is: How is the translocation mechanism
(which is now considered highly conserved among AAA+s) coupled to specific cellular functions? Our long-term
goal is to determine how translocation and unfolding are precisely tuned for different proteostasis and cell stress
response functions. The objective for this application is to identify key allosteric control mechanisms that couple
ATP-driven translocation to substrate recognition, unfolding and degradation. Here we will: (SA1) Determine
mechanisms of protein unfolding and proteolysis by the ClpAP “bacterial proteasome” complex; (SA2) Determine
how Hsp104 interacts with and disaggregates native substrates and amyloids; and (SA3) Determine how the
Hsp70 chaperone collaborates with Hsp104 to promote substrate loading. At the completion of this work we will
identify conformational networks and protein:protein interactions that define how the core translocation cycle
connects allosterically to specify distinct cellular functions of these AAA+ machines.

Terms: <20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><AD dementia><ATP Hydrolysis><Acceleration><Address><Alzheimer Type Dementia><Alzheimer disease dementia><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimers Dementia><Amino Acids><Amyloid><Amyloid Substance><Architecture><Assay><Binding><Bioassay><Biochemical><Biological Assay><Cell Aging><Cell Function><Cell Physiology><Cell Process><Cell Protection><Cell Senescence><Cell Survival><Cell Viability><Cellular Aging><Cellular Function><Cellular Physiology><Cellular Process><Cellular Senescence><Cellular Stress><Cellular Stress Response><Chaperone><Collaborations><Communication><Complex><Coupled><Coupling><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><Cytoprotection><Degenerative Neurologic Disorders><Disease><Disorder><Electron Cryomicroscopy><Engineering / Architecture><Esteroproteases><Event><FRET><Family><Filament><Fluorescence Resonance Energy Transfer><Future><Förster Resonance Energy Transfer><Goals><Grips><Heat shock proteins><Hydrolysis><Life><Macropain><Macroxyproteinase><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mechanics><Mediating><Methods><Mitochondria><Modeling><Molecular Chaperones><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Motor><Multicatalytic Proteinase><NAC precursor><Nature><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><PARK1 protein><PARK4 protein><Paralysis Agitans><Parkinson><Parkinson Disease><Pathway interactions><Peptidases><Peptide Hydrolases><Primary Parkinsonism><Primary Senile Degenerative Dementia><Prosome><Protease Gene><Proteases><Proteasome><Proteasome Endopeptidase Complex><Protein Cleavage><Proteinases><Proteins><Proteolysis><Proteolytic Enzymes><Proteosome><Public Health><Regulatory Protein><Replicative Senescence><Resolution><Rotation><SNCA><SNCA protein><Science><Site><Specific qualifier value><Specified><Stress><Structure><Subcellular Process><Substrate Interaction><Surface><System><Variant><Variation><Work><Yeasts><a-syn><a-synuclein><aberrant protein folding><abnormal protein folding><alpha synuclein><alpha synuclein gene><alphaSP22><aminoacid><amyloid assembly><amyloid formation><asyn><biological adaptation to stress><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><cell stress><conformation><conformational><conformational state><conformationally><conformations><crosslink><cryo-EM><cryoEM><cryogenic electron microscopy><cytoprotective><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><genetic regulatory protein><grasp><human disease><improved><insight><insoluble aggregate><mechanic><mechanical><member><mitochondrial><multicatalytic endopeptidase complex><mutant><neurodegenerative illness><new approaches><non A-beta component of AD amyloid><non A4 component of amyloid precursor><novel approaches><novel strategies><novel strategy><pathologic protein folding><pathway><polypeptide><prevent><preventing><primary degenerative dementia><protein aggregate><protein aggregation><protein homeostasis><protein misfolding><protein protein interaction><proteostasis><reaction; crisis><regulatory gene product><resolutions><senile dementia of the Alzheimer type><single-molecule FRET><single-molecule fluorescence resonance energy transfer><smFRET><stress protein><stress response><stress; reaction><sup35><therapeutic target><unfoldase><yeast prion><α synuclein gene><α-syn><α-synuclein>