Structural Basis for the Formation of PTPRG•CNTN Complexes in Neural Tissues

Title from PDF of title page, viewed on July 22, 2016Dissertation advisor: Samuel BouyainVitaIncludes bibliographical references (pages 112-119)Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2016Receptor protein tyrosine phosphatase gamma (PTPRG) is a cell surface receptor expressed primarily on neurons. It combines cytoplasmic tyrosine phosphatase domains and an extracellular region that includes a carbonic anhydrase-like (CA) domain. This domain mediates binding to members of a family of neural cell adhesion molecules called contactins (CNTNs) that are expressed on neurons during development and adulthood. The ectodomains of CNTNs are organized into six N-terminal immunoglobulin domains followed by four fibronectin type III repeats (FN) and a glycophosphatidylinositol anchor. Previous work demonstrated that PTPRG interacts specifically with CNTN3-6. Here, we combine biochemical and structural approaches to further characterize the interactions between PTPRG and its cognate CNTN partners. In particular, our work indicates that PTPRG associates with CNTN3-6 with similar binding affinities. This finding is consistent with our structural analyses of PTPRG•CNTN3 and PTPRG•CNTN6 complexes suggesting that CNTN3-6 use a conserved interface to bind the CA domain of PTPRG. As a first step to determine the in vivo functions of PTPRG•CNTN complexes, we attempted to localize the sites where these receptors interact. In particular, we identified the PTPRG•CNTN3 complex in the outer segment (OS) of adult mouse retinas. Further investigation of these complexes in the OS revealed that PTPRG and CNTN3 form complexes when expressed on the same cell (cis interactions). However, we also performed cell-aggregation assays indicating that PTPRG and CNTN3 can associate when expressed on distinct cells (trans interactions). To explain how the PTPRG•CNTN3 complex could form in these two distinct geometries, we analyzed the conformations taken by the CNTN3 ectodomain. In particular, our work indicates that the FN1-FN3 of CNTN3 adopts a bent conformation suggesting that the CNTN3 ectodomain bends sharply between FN2 and FN3 domains and then extends in parallel to the cell surface. Importantly, this bent conformation is found in all six CNTN family members suggesting that all CNTNs might lie parallel to the cell membrane. This orientation of CNTN ectodomains would accommodate the formation of cis and trans PTPRG•CNTN complexes. Finally, we discuss the implications of our results in PTPG/CNTN-mediated signaling.Introduction -- Materials and methods -- Structural basis for the conserved interaction of CNTN3, 4, 5 and 6 with the CA domain of PTPRG -- Structural analyses of CNTN ectodomains reveal an unexpected bent conformation -- Final discussion -- Appendi

Altered vacuole membrane protein 1 (VMP1) expression is associated with increased NLRP3 inflammasome activation and mitochondrial dysfunction

International audienceBackground Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson’s disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. Objective This study aimed to characterize how altered VMP1 expression afects NLRP3 infammasome activation and mitochondrial function.Methods VMP1 expression was depleted in a monocytic cell line using CRISPR-Cas9. The efect of VMP1 on NLRP3infammasome activation was examined by stimulating cells with LPS and ATP or α-synuclein fbrils. Infammasome activation was determined by caspase-1 activation using both a FLICA assay and a biosensor as well as by the release of proinfammatory molecules measured by ELISA. RNA-sequencing was utilized to defne global gene expression changes resulting from VMP1 deletion. SERCA activity and mitochondrial function were investigated using various fuorescence microscopy-based approaches including a novel method that assesses the function of individual mitochondria in a cell. Results Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 infammasome activation and release of proinfammatory molecules. Examination of the VMP1-dependent changes in these cells revealed that VMP1 defciency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. Conclusions Collectively, these studies reveal VMP1 as a negative regulator of infammatory responses, and we postulate that decreased expression of VMP1 can aggravate the infammatory sequelae associated with neurodegenerative diseases like PD

Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues.

Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3-6 (CNTN3-6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling

Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues.

Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3-6 (CNTN3-6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential