Inactivation of the KcsA potassium channel explored with heterotetramers

The tetrameric prokaryotic potassium channel KcsA is activated by protons acting on the intracellular aspect of the protein and inactivated through conformational changes in the selectivity filter. Inactivation is modulated by a network of interactions within each protomer between the pore helix and residues at the external entrance of the channel. Inactivation is suppressed by the E71A mutation, which perturbs the stability of this network. Here, cell-free protein synthesis followed by protein purification by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to produce heterotetramers of KcsA that contain different combinations of wild-type and E71A subunits. Single-channel recordings from these heterotetramers reveal how the network of interactions in individual protomers affects ionic conduction and channel inactivation, suggesting that the latter is a cooperative process

Antibody-associated epilepsies: Clinical features, evidence for immunotherapies and future research questions.

PURPOSE: The growing recognition of epilepsies and encephalopathies associated with autoantibodies against surface neuronal proteins (LGI1, NMDAR, CASPR2, GABABR, and AMPAR) means that epileptologists are increasingly asking questions about mechanisms of antibody-mediated epileptogenesis, and about the use of immunotherapies. This review summarizes clinical and paraclinical observations related to autoimmune epilepsies, examines the current evidence for the effectiveness of immunotherapy, and makes epilepsy-specific recommendations for future research. METHOD: Systematic literature search with summary and review of the identified publications. Studies describing the clinical characteristics of autoantibody-associated epilepsies and treatments are detailed in tables. RESULTS: Literature describing the clinical manifestations and treatment of autoimmune epilepsies associated with neuronal cell-surface autoantibodies (NSAbs) is largely limited to retrospective case series. We systematically summarize the features of particular interest to epileptologists dividing patients into those with acute or subacute encephalopathies associated with epilepsy, and those with chronic epilepsy without encephalopathy. Available observational studies suggest that immunotherapies are effective in some clinical circumstances but outcome data collection methods require greater standardization. CONCLUSIONS: The clinical experience captured suggests that clusters of clinical features associate well with specific NSAbs. Intensive and early immunotherapy is indicated when patients present with autoantibody-associated encephalopathies. It remains unclear how patients with chronic epilepsy and the same autoantibodies should be assessed and treated. Tables in this paper provide a comprehensive resource for systematic descriptions of both clinical features and treatments, and highlight limitations of current studies

Development of Novel Methods and their Utilization in the Analysis of the Effect of the N-terminus of Human Protein Arginine Methyltransferase 1 Variant 1 on Enzymatic Activity, Protein-protein Interactions, and Substrate Specificity

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions

Immunotherapy for anti-NMDAR encephalitis: A review of paraneoplastic, autoimmune encephalopathy

[[abstract]]Paraneoplastic immune-mediated encephalopathy is a recently described disease. Dalmau et al first linked the presence of Anti-N-methyl-d-aspartate receptor (anti-NMDAR) antibodies to paraneoplastic psychiatric and neurologic disease after encountering a case of anti-NMDAR encephalitis in a woman with ovarian teratoma in 2007. Nerve cells from a teratoma or previous viral infection could trigger autoantibodies, causing NMDA receptors to become dysfunctional in neurotransmission across synapses. Symptoms of anti-NMDAR encephalitis include prodromal symptoms, psychiatric symptoms, speech dysfunction, seizure, abnormal neurological movement, and autonomic dysfunction. This disease is reversible and treatable; however, early diagnosis and treatment are essential as they may prevent excess antibodies from causing severe or prolonged harm in the brain. First-line Immunotherapy includes intravenous high-dose steroids (methylprednisolone), intravenous immunoglobulin (IVIG), and/or plasmapheresis. Second-line immunotherapy includes targeted B-cell therapy with rituximab and cyclophosphamide. Taiwan is one of the countries where tests for detecting this disease, which are expensive, are not currently available. A cell-based indirect immunofluorescence test for the detection of IgG antibodies against the NMDA receptor should become more available for aiding diagnostics. Most importantly, early immune modulatory therapy including steroid, IVIG, and plasma exchange should become financially more feasible for use in treatment in Taiwan.[[notice]]補正完