Biology subjects

Chiu, J.

Publications and source records attributed to Chiu, J..

4 recordsLinked to original sources

bioRxiv2022

Dynamic Disulfide Bond Topologies in von-Willebrand-Factor's C4-Domain Undermine Platelet Binding

BackgroundThe von Willebrand Factor (vWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large multi-domain mechano-sensitive protein stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the vWF-C4 domain which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. ObjectiveTo quantitatively determine C4s disulfide topologies and their implication in vWFs platelet-binding function via integrin. MethodsWe employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. ResultsWe quantitatively show that two disulfide bonds in the vWF-C4 domain, namely the two major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the RGD-integrin binding motif, and thereby impair integrin-mediated platelet binding. Our combined approach also reveals that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all six vWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. ConclusionOverall, our data put forward a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of vWF with integrin and potentially other partners, thereby critically influencing its hemostatic function. EssentialsO_LIPlatelet integrins interact with the disulfide-bonded C4 domain of von Willebrand Factor C_LIO_LIThe redox state of vWF-C4s disulfide bonds is studied by molecular simulations and experiments C_LIO_LITwo bonds are reduced causing C4 unfolding and disulfide swapping C_LIO_LIOpening of disulfide bonds impairs integrin-mediated platelet binding C_LI

biophysics↗

bioRxiv2022

Mechano-redox control of Mac-1 de-adhesion from ICAM-1 by protein disulfide isomerase promotes directional movement of neutrophils under flow

Macrophage-1 antigen or Mac-1 (CD11b/CD18, M{beta}2) is a leukocyte integrin essential for firm adhesion of neutrophils, lymphocytes and monocytes against flow when recruited to the endothelium. To migrate to the site of inflammation, leukocytes require coordinated adhesion and de-adhesion for directional movement. The vascular thiol isomerase, protein disulfide isomerase (PDI), was found by fluorescence microscopy to colocalize with high affinity Mac-1 at the trailing edge of stimulated neutrophils when adhered to ICAM-1 under fluid shear. From differential cysteine alkylation and mass spectrometry studies, PDI cleaves two allosteric disulfide bonds, C169-C176 and C224-C264, in the {beta}I domain of the {beta}2 subunit, and in mutagenesis and cell transfection studies, cleavage of the C224-C264 disulfide bond was shown to selectively control Mac-1 dis-engagement from ICAM-1 under fluid shear. Molecular dynamics simulations and binding of conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the {beta}I domain that allosterically alters exposure of an I domain epitope and shifts Mac-1 to a lower affinity state. From studies of neutrophil adherence to ICAM-1 under fluid shear, these molecular events promote neutrophil motility in the direction of flow at high shear stress. In summary, shear-dependent PDI cleavage of neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

biochemistry↗

bioRxiv2021

PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock

Temperature compensation is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines (S44, 45 and 47) belonging to the PER phosphodegron, the functional homolog of mammalian PER2s S487 phosphodegron, which impacts temperature compensation. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused decreased temperature compensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the pers phosphocluster decreased thermal compensation, consistent with its inhibitory role on S47 phosphorylation. Interestingly, the S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A resulted in excessive temperature compensation of phosphorylation-dependent PER degradation. Thus, we show a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work also reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock. Author summaryCircadian rhythms are critical adaptive mechanisms that enable most organisms to adjust their physiology and behavior to the changes that occur in their environment every day. Ambient temperature varies constantly, but interestingly molecular circadian pacemakers do not accelerate with increasing temperature, while most biochemical reactions are sensitive to temperature. This phenomenon of circadian temperature compensation is poorly understood. Using genome editing and transgenic approaches, we found that two phosphorylated motifs in the Drosophila PERIOD protein, which regulate stability, impact temperature compensation. Moreover, we observed that mutation of a key Serine residue controlling PER degradation, S47, affects the accumulation of phosphorylated PER in a temperature-dependent manner, and causes PER degradation kinetics to become overly protected from increased temperature. As a result, the circadian clock of S47 mutant flies is excessively temperature-compensated. Our work thus reveals an interesting mechanism that controls temperature compensation in Drosophila. Moreover, comparison with mammals reveal interesting similarities, but also important differences in how temperature compensation of the circadian clock is achieved.

genetics↗

bioRxiv2021

In vitro and in vivo preclinical studies predict REGEN-COV protection against emergence of viral escape in humans

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of minor virus variants in SARS-COV-2 isolates found in COVID-19 patients or identified from preclinical in vitro and in vivo studies. This study demonstrates that a combination of non-competing antibodies, REGEN-COV, not only provides full coverage against current variants of concern/interest but also protects against emergence of new such variants and their potential seeding into the population in a clinical setting.

microbiology↗