Studies on structures, Dynamics and Interactions with small molecules of CNS regeneration Inhibitory components associated with Nogo-A and EphA4

Ph.DDOCTOR OF PHILOSOPH

Intrinsically Unstructured Domain 3 of Hepatitis C Virus NS5A Forms a “Fuzzy Complex” with VAPB-MSP Domain Which Carries ALS-Causing Mutations

Hepatitis C virus (HCV) affects nearly 200 million people worldwide and is a leading factor for serious chronic liver diseases. For replicating HCV genome, the membrane-associated replication machinery needs to be formed by both HCV non-structural proteins including NS5A and human host factors. Recently NS5A has been identified to bind ER-anchored human VAP proteins and consequently this interaction may serve as a novel target for design of anti-HCV drugs. So far no biophysical characterization of this interaction has been reported. Here, we dissected the 243-residue VAPB into 4 and 447-residue NS5A into 10 fragments, followed by CD and NMR characterization of their structural properties. Subsequently, binding interactions between these fragments have been extensively assessed by NMR HSQC titration which is very powerful in detecting even very weak binding. The studies lead to three important findings: 1). a “fuzzy complex” is formed between the intrinsically-unstructured third domain (D3) of NS5A and the well-structured MSP domain of VAPB, with an average dissociation constant (Kd) of ∼5 µM. 2). The binding-important residues on both NS5A-D3 and VAPB-MSP have been successfully mapped out, which provided experimental constraints for constructing the complex structure. In the complex, unstructured D3 binds to three surface pockets on one side of the MSP structure. Interestingly, two ALS-causing mutations T46I and P56S are also located on the D3-MSP interface. Moreover, NS5A-D3, FFAT-containing proteins and EphA4 appear to have overlapped binding interfaces on the MSP domain. 3). NS5A-D3 has been experimentally confirmed to competes with EphA4 in binding to the MSP domain, and T46I mutation of MSP dramatically abolishes its binding ability to D3. Our study not only provides essential foundation for further deciphering structure and function of the HCV replication machinery, but may also shed light on rationalizing a recent observation that a chronic HCV patient surprisingly developed ALS-like syndrome

Identification of New Glutamate Decarboxylases from \u3cem\u3eStreptomyces\u3c/em\u3e for Efficient Production of γ-Aminobutyric Acid in Engineered \u3cem\u3eEscherichia coli\u3c/em\u3e

Background Gamma (γ)-Aminobutyric acid (GABA) as a bioactive compound is used extensively in functional foods, pharmaceuticals and agro-industry. It can be biosynthesized via decarboxylation of monosodium glutamate (MSG) or L-glutamic acid (L-Glu) by glutamate decarboxylase (GAD; EC4.1.1.15). GADs have been identified from a variety of microbial sources, such as Escherichia coli and lactic acid bacteria. However, no GADs from Streptomyces have been characterized. The present study is aimed to identify new GADs from Streptomyces strains and establish an efficient bioproduction platform for GABA in E. coli using these enzymes. Results By sequencing and analyzing the genomes of three Streptomycesstrains, three putative GADs were discovered, including StGAD from Streptomyces toxytricini NRRL 15443, SsGAD from Streptomyces sp.MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982. The corresponding genes were cloned from these strains and heterologously expressed in E. coli BL21(DE3). The purified GAD proteins showed a similar molecular mass to GadB from E. coliBL21(DE3). The optimal reaction temperature is 37 °C for all three enzymes, while the optimum pH values for StGAD, SsGAD and ScGAD are 5.2, 3.8 and 4.2, respectively. The kinetic parameters including Vmax, Km, kcat and kcat/Km values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with kcat/Km values of 0.62 and 1.21 mM− 1·s− 1, respectively. In addition, engineered E. coli strains harboring StGAD, SsGAD and ScGAD were used as whole-cell biocatalysts for production of GABA from L-Glu. E. coli/SsGAD showed the highest capability of GABA production. The cells were repeatedly used for 10 times, with an accumulated yield of 2.771 kg/L and an average molar conversion rate of 67% within 20 h. Conclusions Three new GADs have been functionally characterized from Streptomyces, among which two showed higher catalytic efficiency than previously reported GADs. Engineered E. coli harboring SsGAD provides a promising cost-effective bioconversion system for industrial production of GABA

VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B

10.1371/journal.pone.0040341PLoS ONE77

A small molecule agonist of EphA2 receptor tyrosine kinase inhibits tumor cell migration in vitro and prostate cancer metastasis in vivo

10.1371/journal.pone.0042120PLoS ONE78

Structural, Stability, Dynamic and Binding Properties of the ALS-Causing T46I Mutant of the hVAPB MSP Domain as Revealed by NMR and MD Simulations

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

10.1371/journal.pone.0074040PLoS ONE89-POLN

Learned Smartphone ISP on Mobile GPUs with Deep Learning, Mobile AI & AIM 2022 Challenge: Report

The role of mobile cameras increased dramatically over the past few years, leading to more and more research in automatic image quality enhancement and RAW photo processing. In this Mobile AI challenge, the target was to develop an efficient end-to-end AI-based image signal processing (ISP) pipeline replacing the standard mobile ISPs that can run on modern smartphone GPUs using TensorFlow Lite. The participants were provided with a large-scale Fujifilm UltraISP dataset consisting of thousands of paired photos captured with a normal mobile camera sensor and a professional 102MP medium-format FujiFilm GFX100 camera. The runtime of the resulting models was evaluated on the Snapdragon's 8 Gen 1 GPU that provides excellent acceleration results for the majority of common deep learning ops. The proposed solutions are compatible with all recent mobile GPUs, being able to process Full HD photos in less than 20-50 milliseconds while achieving high fidelity results. A detailed description of all models developed in this challenge is provided in this paper

"Dark Mediators" of Proteins as Revealed by NMR in Water: Residue-selective Anion Bindings that are Masked by Pre-existing Buffer

Ions are commonly believed to impose their effects on proteins by unspecific electrostatic screening. Here, by NMR we reveal that in water sulfate, chloride and thiocyanate are able to bind a well-folded WW domain at distinctive residues and affinities, which is surprisingly masked by the pre-existing buffer. Our study reveals that the specific anion binding is so ubiquitous and consequently no longer negligible in establishing "postreductionist framework" for protein biochemistry