Biology subjects

Gestwicki, J.

Publications and source records attributed to Gestwicki, J..

5 recordsLinked to original sources

bioRxiv2021

The YPhi Motif Defines the Structure-Activity Relationships of Human 20S Proteasome Activators

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesized and screened [~]120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (Y{Phi}) motif. To gain further insight, we created a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A.A cryo-EM structure of PA26E102A-h20S identified key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrated that the Y{Phi} sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

biochemistry↗

bioRxiv2020

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Over-expression and aggregation of -synuclein (ASyn) are linked to the onset and pathology of Parkinsons disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.

neuroscience↗

bioRxiv2020

Cytoskeletal tension forces mitohormesis

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programmed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of ageing. However, the physical characteristics of the extracellular matrix are also altered in cancer and in aging tissues. We demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via SLC9A1-dependent ion exchange and HSF1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/979583v3_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1ecdaceorg.highwire.dtl.DTLVardef@1112c32org.highwire.dtl.DTLVardef@6c99a1org.highwire.dtl.DTLVardef@1da1e5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy

Dominant mutations in the HSP70 co-chaperone DNAJB6 cause a late onset muscle disease termed limb girdle muscular dystrophy type 1D (LGMD1D), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMD1D, we find that the toxicity associated with disease-associated DNAJB6 requires its interaction with HSP70, and that abrogating this interaction genetically or with small molecules is protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffuses rapidly between the Z-disc and sarcoplasm, the rate of HSP70s diffusion in LGMD1D mouse muscle is diminished likely because it has an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMD1D mice with a small molecule inhibitor of the DNAJ-HSP70 complex re-mobilizes HSP70, improves strength and corrects myopathology. These data support a model in which LGMD1D mutations in DNAJB6 are a gain-of-function disease that is, counter-intuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70:DNAJB6 may be effective in treating this inherited muscular dystrophy.

cell biology↗

bioRxiv2017

The small molecule KHS101 induces bioenergetic dysfunction in glioblastoma cells through inhibition of mitochondrial HSPD1

Pharmacological inhibition of uncontrolled cell growth with small molecule inhibitors is a potential strategy against glioblastoma multiforme (GBM), the most malignant primary brain cancer. Phenotypic profiling of the neurogenic small molecule KHS101 revealed the chemical induction of lethal cellular degradation in molecularly-diverse GBM cells, independent of their tumor subtype, whereas non-cancerous brain cells remained viable. Mechanism-of-action (MOA) studies showed that KHS101 specifically bound and inhibited the mitochondrial chaperone HSPD1. In GBM but not non-cancerous brain cells, KHS101 elicited the aggregation of an enzymatic network that regulates energy metabolism. Compromised glycolysis and oxidative phosphorylation (OXPHOS) resulted in the metabolic energy depletion in KHS101-treated GBM cells. Consistently, KHS101 induced key mitochondrial unfolded protein response factor DDIT3 in vitro and in vivo, and significantly reduced intracranial GBM xenograft tumor growth upon systemic administration, without discernible side effects. These findings suggest targeting of HSPD1-dependent oncometabolic pathways as an anti-GBM therapy.

cancer biology↗