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Zhao, Y. J.

Publications and source records attributed to Zhao, Y. J..

4 recordsLinked to original sources

Pyruvate kinase activates SARM1 to exacerbate axonal degeneration in diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is a prevalent and disabling complication of diabetes, characterized by progressive axonal degeneration. However, the molecular link between hyperglycemia and axon injury remains unclear. Here, we identify pyruvate kinase M (PKM) as a direct metabolic activator of the NADase SARM1 under high-glucose conditions. Proteomic and biochemical analyses reveal that PKM binds the TIR domain of SARM1 via its C-terminal region, allosterically enhancing NADase activity independently of PKMs glycolytic role. In dorsal root ganglion (DRG) neurons, hyperglycemia strengthens the PKM-SARM1 interaction, driving NAD depletion, axonal fragmentation, and sensory dysfunction. Genetic depletion of PKM protects against streptozotocin-induced neuropathy, preserving nerve fiber density, restoring NAD levels, and alleviating mechanical allodynia. Most notably, we developed Pep-SP1, a competitive inhibitory peptide derived from residues 645-655 of the SARM1 TIR domain, which selectively disrupts the PKM-SARM1 interaction without impairing PKM metabolism or SARM1 catalytic activity. Systemic delivery of Pep-SP1 attenuates axonal degeneration and improves sensory outcomes in diabetic mice. By targeting a disease-potentiating interface, we expand therapeutic strategies beyond catalytic and allosteric inhibition, offering a mechanistically distinct avenue for axon protection with broad relevance to metabolic and neurodegenerative disorders.

neuroscience↗

A Conformation-specific Nanobody Targeting the NMN-activated State of SARM1

Upon axonal injury, Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is activated by nicotinamide mononucleotide (NMN) to deplete NAD and consequently promote the process of axon degeneration (AxD). Currently, only the inactive form of SARM1 in its auto-inhibitory conformation has been resolved. The flexibility of the enzymatically active form of SARM1 has so far precluded its structural determination. To solve the problem, we generated a stabilizing nanobody, Nb-C6, that specifically recognized only the NMN-activated form of SARM1. The conformation specificity was verified by immunoprecipitation and surface plasmon resonance. Fluorescently labeled Nb-C6 could immunostain only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN. Expression of Nb-C6 in live cells resulted in stabilization of the active form of the endogenous and exogenous SARM1, producing and elevating cellular levels of cyclic ADP-ribose, a calcium messenger. Cryo-EM of the NMN-activated SARM1 complexed by Nb-C6 showed an octameric structure resembling a "blooming lotus" with the ARM domains bending significantly inward and swinging out together with the TIR domains to form the "petals of the lotus". Nb-C6 bound to the SAM domain of the activated SARM1 and stabilized its Armadillo repeat motif domain. Analyses using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and cross-linking MS (XL-MS) indicate that the activated SARM1 is highly dynamic and flexible and the neighboring TIRs form dimers via the surface close to one BB loop. The Nanobody is thus a valuable tool for delineating the mechanism of activation of SARM1 in AxD and other cellular processes.

molecular biology↗

Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red-shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine as a covalent inhibitor of SARM1 that reacted with Cys311 in its Armadillo-domain and blocked its NMN-activation, protecting axons from degeneration. CryoEM showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering an unsuspected neuroprotective mechanism of dihydropyridines.

biochemistry↗

Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease has been implicated in virus maturation, dysregulation of host inflammation and antiviral immune responses. We showed that PLpro preferably cleaves the K48-ubiquitin linkage while also being capable of cleaving ISG15 modification. The multiple functions of PLpro render it a promising drug target. Therefore, we screened an FDA-approved drug library and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising IC50 of 2.1 M. The co-crystal structure of SARS-CoV-2 PLpro-C111S in complex with GRL0617 suggests that GRL0617 is a non-covalent inhibitor. NMR data indicate that GRL0617 blocks the binding of ISG15 to PLpro. The antiviral activity of GRL0617 reveal that PLpro is a promising drug target for therapeutically treating COVID-19. One Sentence SummaryCo-crystal structure of PLpro in complex with GRL0617 reveals the druggability of PLpro for SARS-CoV-2 treatment.

biochemistry↗