Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding.

BACKGROUND: The 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 that is 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. OBJECTIVE: To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF's platelet binding function. METHODS: We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. RESULTS: We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 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 integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal 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 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. CONCLUSIONS: Our data suggests 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.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces ex vivo and in vivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces.

Compartmentalization of Total and Virus-Specific Tissue-Resident Memory CD8+ T Cells in Human Lymphoid Organs.

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections.

Abnormal glucocorticoid receptor mRNA and protein isoform expression in the prefrontal cortex in psychiatric illness.

Stress has been implicated in the onset and illness course of schizophrenia and bipolar disorder. The effects of stress in these disorders may be mediated by abnormalities of the hypothalamic-pituitary-adrenal axis, and its corticosteroid receptors. We investigated mRNA expression of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), and protein expression of multiple GRα isoforms, in the prefrontal cortex of 37 schizophrenia cases and 37 matched controls. Quantitative real-time PCR, western blotting, and luciferase assays were employed. In multiple regression analysis, schizophrenia diagnosis was a significant predictor of total GR mRNA expression (p<0.05), which was decreased (11.4%) in schizophrenia cases relative to controls. No significant effect of diagnosis on MR mRNA was detected. At the protein level, no significant predictors of total GRα protein or the full-length GRα isoform were identified. However, schizophrenia diagnosis was a strong predictor (p<0.0005) of the abundance of a truncated ≈ 50 kDa GRα protein isoform, putative GRα-D1, which was increased in schizophrenia cases (80.4%) relative to controls. This finding was replicated in a second cohort of 35 schizophrenia cases, 34 bipolar disorder cases, and 35 controls, in which both schizophrenia and bipolar disorder diagnoses were significant predictors of putative GRα-D1 abundance (p<0.05 and p=0.005, respectively). Full-length GRα was increased in bipolar disorder relative to schizophrenia cases. Luciferase assays demonstrated that the GRα-D1 isoform can activate transcription at glucocorticoid response elements. These findings confirm total GR mRNA reductions in schizophrenia and provide the first evidence of GR protein isoform abnormalities in schizophrenia and bipolar disorder.

Full length TrkB potentiates estrogen receptor alpha mediated transcription suggesting convergence of susceptibility pathways in schizophrenia.

In this study, we determined if estrogen receptor alpha (ERα) can interact with the full length tropomyosin receptor kinase B (TrkB-TK+), both of which are implicated in schizophrenia pathogenesis. Using neuronal (SHSY5Y) and non-neuronal (CHOK1) cell-lines, we showed that TrkB-TK+ can increase transcription at estrogen response elements (EREs) with and without exogenous estrogen treatment. In the presence of estrogen, TrkB-TK+ further potentiated the effect of estrogen stimulation on ERα-mediated transcription. This synergistic effect of TrkB-TK+ on ERα-mediated transcription was not due to direct effects of TrkB-TK+ in the nucleus, but occurred through cytoplasmic signaling of TrkB-TK+ via the MAPK/ERK pathway to phosphorylate ERα, leading to an induction in ERα-mediated transcription. When we examined the PI3K/AKT pathway, we found that PI3K/AKT activity constitutively inhibited baseline transcription at EREs. Furthermore, we showed that signaling via PI3K/AKT inhibited TrkB-TK+-dependent transcriptional potentiation at EREs. Our findings suggest that TrkB-TK+-linked second messenger signaling pathways can reciprocally regulate ERα-mediated transcription at EREs. Considering that both ERα and TrkB-TK+ expression are reduced in schizophrenia, our findings suggest that dysfunction in TrkB-TK+ signaling may occur upstream of, or in conjunction with a dysfunction in ERα, and that transcriptional regulation by ERα may be decreased by reductions in TrkB-TK+.

Identification of putative functional motifs in viral proteins essential for human cytomegalovirus DNA replication.

Six of the eleven genes essential for Human cytomegalovirus (HCMV) DNA synthesis have been analyzed for putative structural motifs that may have a significant functional role in DNA replication. The genes studied encode for the DNA polymerase accessory protein (UL44), single-stranded DNA binding protein (UL57), primase-helicase complex (UL70, UL102, and UL105), and the putative initiator protein (UL84). The full-length open reading frames of these genes were highly conserved between ten isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. Interesting motifs identified include casein kinase-2 (CKII) phosphorylation sites, a microbodies signal motif in UL57, and an ATP binding site in the putative UL105 helicase. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein and identified cysteine motifs that have potential implications for UL57 and UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation, and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes.