Hodgkin-Huxley-Katz Prize Lecture: Genetic and pharmacological control of glutamate receptor channel through a highly conserved gating motif.

Glutamate receptors are essential ligand-gated ion channels in the central nervous system that mediate excitatory synaptic transmission in response to the release of glutamate from presynaptic terminals. The structural and biophysical basis underlying the function of these receptors has been studied for decades by a wide range of approaches. However recent structural, pharmacological and genetic studies have provided new insight into the regions of this protein that are critical determinants of receptor function. Lack of variation in specific areas of the protein amino acid sequences in the human population has defined three regions in each receptor subunit that are under selective pressure, which has focused research efforts and driven new hypotheses. In addition, these three closely positioned elements reside near a cavity that is shown by multiple studies to be a likely site of action for allosteric modulators, one of which is currently in use as an FDA-approved anticonvulsant. These structural elements are capable of controlling gating of the pore, and appear to permit some modulators bound within the cavity to also alter permeation properties. This creates a new precedent whereby features of the channel pore can be modulated by exogenous drugs that bind outside the pore. The convergence of structural, genetic, biophysical and pharmacological approaches is a powerful means to gain insight into the complex biological processes defined by neurotransmitter receptor function.

Phospholamban interactome in cardiac contractility and survival: A new vision of an old friend.

Depressed sarcoplasmic reticulum (SR) calcium cycling, reflecting impaired SR Ca-transport and Ca-release, is a key and universal characteristic of human and experimental heart failure. These SR processes are regulated by multimeric protein complexes, including protein kinases and phosphatases as well as their anchoring and regulatory subunits that fine-tune Ca-handling in specific SR sub-compartments. SR Ca-transport is mediated by the SR Ca-ATPase (SERCA2a) and its regulatory phosphoprotein, phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA2a and phosphorylation by protein kinase A (PKA) or calcium-calmodulin-dependent protein kinases (CAMKII) relieves these inhibitory effects. Recent studies identified additional regulatory proteins, associated with PLN, that control SR Ca-transport. These include the inhibitor-1 (I-1) of protein phosphatase 1 (PP1), the small heat shock protein 20 (Hsp20) and the HS-1 associated protein X-1 (HAX1). In addition, the intra-luminal histidine-rich calcium binding protein (HRC) has been shown to interact with both SERCA2a and triadin. Notably, there is physical and direct interaction between these protein players, mediating a fine-cross talk between SR Ca-uptake, storage and release. Importantly, regulation of SR Ca-cycling by the PLN/SERCA interactome does not only impact cardiomyocyte contractility, but also survival and remodeling. Indeed, naturally occurring variants in these Ca-cycling genes modulate their activity and interactions with other protein partners, resulting in depressed contractility and accelerated remodeling. These genetic variants may serve as potential prognostic or diagnostic markers in cardiac pathophysiology.

A De Novo Sequence Variant in Barrier-to-Autointegration Factor Is Associated with Dominant Motor Neuronopathy.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor-Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.

Infection outcome needs two to tango: human host and the pathogen.

Infectious diseases are potential drivers for human evolution, through a complex, continuous and dynamic interaction between the host and the pathogen/s. It is this dynamic interaction that contributes toward the clinical outcome of a pathogenic disease. These are modulated by contributions from the human genetic variants, transcriptional response (including noncoding RNA) and the pathogen's genome architecture. Modern genomic tools and techniques have been crucial for the detection and genomic characterization of pathogens with respect to the emerging infectious diseases. Aided by next-generation sequencing (NGS), risk stratification of host population/s allows for the identification of susceptible subgroups and better disease management. Nevertheless, many challenges to a general understanding of host-pathogen interactions remain. In this review, we elucidate how a better understanding of the human host-pathogen interplay can substantially enhance, and in turn benefit from, current and future applications of multi-omics based approaches in infectious and rare diseases. This includes the RNA-level response, which modulates the disease severity and outcome. The need to understand the role of human genetic variants in disease severity and clinical outcome has been further highlighted during the Coronavirus disease 2019 (COVID-19) pandemic. This would enhance and contribute toward our future pandemic preparedness.

Phosphorylation of T897 in the dimerization domain of Gemin5 modulates protein interactions and translation regulation.

Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent functions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular proteins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2-3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12-13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and currently known pathogenic variants indicates a lack of overlapping of the affected residues within the functional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation.

The Impact of Rare Human Variants on Barrier-To-Auto-Integration Factor 1 (Banf1) Structure and Function.

Barrier-to-Autointegration Factor 1 (Banf1/BAF) is a critical component of the nuclear envelope and is involved in the maintenance of chromatin structure and genome stability. Banf1 is a small DNA binding protein that is conserved amongst multicellular eukaryotes. Banf1 functions as a dimer, and binds non-specifically to the phosphate backbone of DNA, compacting the DNA in a looping process. The loss of Banf1 results in loss of nuclear envelope integrity and aberrant chromatin organisation. Significantly, mutations in Banf1 are associated with the severe premature ageing syndrome, Néstor-Guillermo Progeria Syndrome. Previously, rare human variants of Banf1 have been identified, however the impact of these variants on Banf1 function has not been explored. Here, using in silico modelling, biophysical and cell-based approaches, we investigate the effect of rare human variants on Banf1 structure and function. We show that these variants do not significantly alter the secondary structure of Banf1, but several single amino acid variants in the N- and C-terminus of Banf1 impact upon the DNA binding ability of Banf1, without altering Banf1 localisation or nuclear integrity. The functional characterisation of these variants provides further insight into Banf1 structure and function and may aid future studies examining the potential impact of Banf1 function on nuclear structure and human health.

Towards precision medicine: advances in computational approaches for the analysis of human variants.

Variations and similarities in our individual genomes are part of our history, our heritage, and our identity. Some human genomic variants are associated with common traits such as hair and eye color, while others are associated with susceptibility to disease or response to drug treatment. Identifying the human variations producing clinically relevant phenotypic changes is critical for providing accurate and personalized diagnosis, prognosis, and treatment for diseases. Furthermore, a better understanding of the molecular underpinning of disease can lead to development of new drug targets for precision medicine. Several resources have been designed for collecting and storing human genomic variations in highly structured, easily accessible databases. Unfortunately, a vast amount of information about these genetic variants and their functional and phenotypic associations is currently buried in the literature, only accessible by manual curation or sophisticated text text-mining technology to extract the relevant information. In addition, the low cost of sequencing technologies coupled with increasing computational power has enabled the development of numerous computational methodologies to predict the pathogenicity of human variants. This review provides a detailed comparison of current human variant resources, including HGMD, OMIM, ClinVar, and UniProt/Swiss-Prot, followed by an overview of the computational methods and techniques used to leverage the available data to predict novel deleterious variants. We expect these resources and tools to become the foundation for understanding the molecular details of genomic variants leading to disease, which in turn will enable the promise of precision medicine.