Multimerization of TREM2 is impaired by Alzheimer's disease-associated variants.

INTRODUCTION: The immune receptor triggering receptor expressed on myeloid cells 2 (TREM2) is among the strongest genetic risk factors for Alzheimer's disease (AD) and is a therapeutic target. TREM2 multimers have been identified in crystallography and implicated in the efficacy of antibody therapeutics; however, the molecular basis for TREM2 multimerization remains poorly understood. METHODS: We used molecular dynamics simulations and binding energy analysis to determine the effects of AD-associated variants on TREM2 multimerization and validated with experimental results. RESULTS: TREM2 trimers remained stably bound, driven primarily by salt bridge between residues D87 and R76 at the interface of TREM2 units. This salt bridge was disrupted by the AD-associated variants R47H and R98W and nearly ablated by the D87N variant. This decreased binding among TREM2 multimers was validated with co-immunoprecipitation assays. DISCUSSION: This study uncovers a molecular basis for TREM2 forming stable trimers and unveils a novel mechanism by which TREM2 variants may increase AD risk by disrupting TREM2 oligomerization to impair TREM2 normal function. HIGHLIGHTS: Triggering receptor expressed on myeloid cells 2 (TREM2) multimerization could regulate TREM2 activation and function. D87-R76 salt bridges at the interface of TREM2 units drive the formation of stable TREM2 dimers and trimers. Alzheimer's disease (AD)-associated R47H and R98W variants disrupt the D87-R76 salt bridge. The AD-associated D87N variant leads to complete loss of the D87-R76 salt bridge.

Electronegative clusters modulate folding status and RNA binding of unstructured RNA-binding proteins.

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA-binding affinity of SLBP to the sub-picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA-bound resembling state. We find that in the RNA-bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA-binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double-stranded RNA-binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA-binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface.

Vitamin D3 and its hydroxyderivatives as promising drugs against COVID-19: a computational study.

The epidemiologic correlation between the poor prognosis of SARS-CoV-2 infection and vitamin D deficiency has been observed worldwide, however, their molecular mechanisms are not fully understood. In this study, we used combined molecular docking, molecular dynamics simulations and binding free energy analyses to investigate the potentials of vitamin D3 and its hydroxyderivatives as TMPRSS2 inhibitor and to inhibit the SARS-CoV-2 receptor binding domain (RBD) binding to angiotensin-converting enzyme 2 (ACE2), as well as to unveil molecular and structural basis of 1,25(OH)2D3 capability to inhibit ACE2 and SARS-CoV-2 RBD interactions. The results show that vitamin D3 and its hydroxyderivatives are favorable to bind active site of TMPRSS2 and the binding site(s) between ACE2 and SARS-CoV2-RBD, which indicate that vitamin D3 and its biologically active hydroxyderivatives can serve as TMPRSS2 inhibitor and can inhibit ACE2 binding of SARS-CoV-2 RBD to prevent SARS-CoV-2 entry. Interaction of 1,25(OH)2D3 with SARS-CoV-2 RBD and ACE2 resulted in the conformation and dynamical motion changes of the binding surfaces between SARS-CoV-2 RBD and ACE2 to interrupt the binding of SARS-CoV-2 RBD with ACE2. The interaction of 1,25(OH)2D3 with TMPRSS2 also caused the conformational and dynamical motion changes of TMPRSS2, which could affect TMPRSS2 to prime SARS-CoV-2 spike proteins. Our results propose that vitamin D3 and its biologically active hydroxyderivatives are promising drugs or adjuvants in the treatment of COVID-19. Communicated by Ramaswamy H. Sarma.

In silico identification of available drugs targeting cell surface BiP to disrupt SARS-CoV-2 binding and replication: Drug repurposing approach.

AIMS: Cell surface binding immunoglobin protein (csBiP) is predicted to be susceptible to SARS-CoV-2 binding. With a substrate-binding domain (SBD) that binds to polypeptides and a nucleotide-binding domain (NBD) that can initiate extrinsic caspase-dependent apoptosis, csBiP may be a promising therapeutic target for COVID-19. This study aims to identify FDA-approved drugs that can neutralize viral binding and prevent viral replication by targeting the functional domains of csBiP. METHODS: In silico screening of 1999 FDA-approved drugs against the functional domains of BiP were performed using three molecular docking programs to avoid bias from individual docking programs. Top ligands were selected by averaging the ligand rankings from three programs. Interactions between top ligands and functional domains of BiP were analyzed. KEY FINDINGS: The top 10 SBD-binding candidates are velpatasvir, irinotecan, netupitant, lapatinib, doramectin, conivaptan, fenoverine, duvelisib, irbesartan, and pazopanib. The top 10 NBD-binding candidates are nilotinib, eltrombopag, grapiprant, topotecan, acetohexamide, vemurafenib, paritaprevir, pixantrone, azosemide, and piperaquine-phosphate. Among them, Velpatasvir and paritaprevir are antiviral agents that target the protease of hepatitis C virus. Netupitant is an anti-inflammatory drug that inhibits neurokinin-1 receptor, which contributes to acute inflammation. Grapiprant is an anti-inflammatory drug that inhibits the prostaglandin E2 receptor protein subtype 4, which is expressed on immune cells and triggers inflammation. These predicted SBD-binding drugs could disrupt SARS-CoV-2 binding to csBiP, and NBD-binding drugs may falter viral attachment and replication by locking the SBD in closed conformation and triggering apoptosis in infected cells. SIGNIFICANCE: csBiP appears to be a novel therapeutic target against COVID-19 by preventing viral attachment and replication. These identified drugs could be repurposed to treat COVID-19 patients.

LAT1 Protein Content Increases Following 12 Weeks of Resistance Exercise Training in Human Skeletal Muscle.

Introduction: Amino acid transporters are essential for cellular amino acid transport and promoting protein synthesis. While previous literature has demonstrated the association of amino acid transporters and protein synthesis following acute resistance exercise and amino acid supplementation, the chronic effect of resistance exercise and supplementation on amino acid transporters is unknown. The purpose herein was to determine if amino acid transporters and amino acid metabolic enzymes were related to skeletal muscle hypertrophy following resistance exercise training with different nutritional supplementation strategies. Methods: 43 college-aged males were separated into a maltodextrin placebo (PLA, n = 12), leucine (LEU, n = 14), or whey protein concentrate (WPC, n = 17) group and underwent 12 weeks of total-body resistance exercise training. Each group's supplement was standardized for total energy and fat, and LEU and WPC supplements were standardized for total leucine (6 g/d). Skeletal muscle biopsies were obtained prior to training and ~72 h following each subject's last training session. Results: All groups increased type I and II fiber cross-sectional area (fCSA) following training (p < 0.050). LAT1 protein increased following training (p < 0.001) and increased more in PLA than LEU and WPC (p < 0.050). BCKDHα protein increased and ATF4 protein decreased following training (p < 0.001). Immunohistochemistry indicated total LAT1/fiber, but not membrane LAT1/fiber, increased with training (p = 0.003). Utilizing all groups, the change in ATF4 protein, but no other marker, trended to correlate with the change in fCSA (r = 0.314; p = 0.055); however, when regression analysis was used to delineate groups, the change in ATF4 protein best predicted the change in fCSA only in LEU (r 2 = 0.322; p = 0.043). In C2C12 myoblasts, LAT1 protein overexpression caused a paradoxical decrease in protein synthesis levels (p = 0.002) and decrease in BCKDHα protein (p = 0.001). Conclusions: Amino acid transporters and metabolic enzymes are affected by resistance exercise training, but do not appear to dictate muscle fiber hypertrophy. In fact, overexpression of LAT1 in vitro decreased protein synthesis.

Skeletal muscle mitochondrial volume and myozenin-1 protein differences exist between high versus low anabolic responders to resistance training.

BACKGROUND: We sought to examine how 12 weeks of resistance exercise training (RET) affected skeletal muscle myofibrillar and sarcoplasmic protein levels along with markers of mitochondrial physiology in high versus low anabolic responders. METHODS: Untrained college-aged males were classified as anabolic responders in the top 25th percentile (high-response cluster (HI); n = 13, dual x-ray absorptiometry total body muscle mass change (Δ) = +3.1 ± 0.3 kg, Δ vastus lateralis (VL) thickness = +0.59 ± 0.05 cm, Δ muscle fiber cross sectional area = +1,426 ± 253 µm2) and bottom 25th percentile (low-response cluster (LO); n = 12, +1.1 ± 0.2 kg, +0.24 ± 0.07 cm, +5 ± 209 µm2; p < 0.001 for all Δ scores compared to HI). VL muscle prior to (PRE) and following RET (POST) was assayed for myofibrillar and sarcoplasmic protein concentrations, myosin and actin protein content, and markers of mitochondrial volume. Proteins related to myofibril formation, as well as whole lysate PGC1-α protein levels were assessed. RESULTS: Main effects of cluster (HI > LO, p = 0.018, Cohen's d = 0.737) and time (PRE > POST, p = 0.037, Cohen's d = -0.589) were observed for citrate synthase activity, although no significant interaction existed (LO PRE = 1.35 ± 0.07 mM/min/mg protein, LO POST = 1.12 ± 0.06, HI PRE = 1.53 ± 0.11, HI POST = 1.39 ± 0.10). POST myofibrillar myozenin-1 protein levels were up-regulated in the LO cluster (LO PRE = 0.96 ± 0.13 relative expression units, LO POST = 1.25 ± 0.16, HI PRE = 1.00 ± 0.11, HI POST = 0.85 ± 0.12; within-group LO increase p = 0.025, Cohen's d = 0.691). No interactions or main effects existed for other assayed markers. DISCUSSION: Our data suggest myofibrillar or sarcoplasmic protein concentrations do not differ between HI versus LO anabolic responders prior to or following a 12-week RET program. Greater mitochondrial volume in HI responders may have facilitated greater anabolism, and myofibril myozenin-1 protein levels may represent a biomarker that differentiates anabolic responses to RET. However, mechanistic research validating these hypotheses is needed.