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Deletion of the protein-protein interaction SH3 domain of the membrane remodeling amphiphysin 2 (BIN1) protein was found to lead to centronuclear myopathy in patients, yet only few interaction partners of BIN1 SH3 have been identified so far, precluding a better understanding of the pathomechanism. Here we used the holdup assay to proteome-wide measure steady-state affinity constants of BIN1 SH3 domain for thousands of full-length cellular proteins, as well as for hundreds of putative SH3-binding sites found within the identified BIN1 partners. Besides confirming known partners, such as dynamin 2 (DNM2), we also identified and affinity-characterized numerous others, like SMCHD1, which were previously implicated in different neuromuscular disorders. We also assessed the impact of a set of rare natural BIN1 SH3 domain variants on affinity interactomes and identified potentially harmful ones that exhibited perturbed affinity profiles, whose impacts were confirmed in a cellular assay for BIN1-mediated membrane remodeling, tentatively connecting them to neuromuscular disorders. In the study, we develop a new affinity-interactomic strategy, which can be generally applied to study the consequences of disease-associated genomic variants of any kind.

Significance Membrane remodeling and trafficking is essential for intracellular organization under normal conditions and can be altered in a plethora of diseases. Here we characterized the action of amphiphysin (BIN1) and dynamin (DNM2), two main regulators of membrane remodeling mutated in congenital myopathies. We found their interplay is necessary for membrane fission in vitro and to maintain muscle homeostasis in vivo. Moreover, increasing BIN1 expression was validated as a therapeutic approach to ameliorate both mild and severe forms of DNM2-associated myopathies in mice.

Dynamin 2 mechanoenzyme is a key regulator of membrane remodeling and gain-of-function mutations in its gene cause centronuclear myopathies. Here, we investigate the functions of dynamin 2 isoforms and their associated phenotypes and, specifically, the ubiquitous and muscle-specific dynamin 2 isoforms expressed in skeletal muscle. In cell-based assays, we show that a centronuclear myopathy-related mutation in the ubiquitous but not the muscle-specific dynamin 2 isoform causes increased membrane fission. In vivo, overexpressing the ubiquitous dynamin 2 isoform correlates with severe forms of centronuclear myopathy, while overexpressing the muscle-specific isoform leads to hallmarks seen in milder cases of the disease. Previous mouse studies suggested that reduction of the total dynamin 2 pool could be therapeutic for centronuclear myopathies. Here, dynamin 2 splice switching from muscle-specific to ubiquitous dynamin 2 aggravated the phenotype of a severe X-linked form of centronuclear myopathy caused by loss-of-function of the MTM1 phosphatase, supporting the importance of targeting the ubiquitous isoform for efficient therapy in muscle. Our results highlight that the ubiquitous and not the muscle-specific dynamin 2 isoform is the main modifier contributing to centronuclear myopathy pathology.

International audience; Objective: Glycogen storage diseases (GSDs) are severe human disorders resulting from abnormal glucose metabolism, and all previously described GSDs segregate as autosomal recessive or X-linked traits. In this study, we aimed to molecularly characterize the first family with a dominant GSD. Methods: We describe a dominant GSD family with 13 affected members presenting with adult-onset muscle weakness, and we provide clinical, metabolic, histological, and ultrastructural data. We performed exome sequencing to uncover the causative gene, and functional experiments in the cell model and on recombinant proteins to investigate the pathogenic effect of the identified mutation. Results: We identified a heterozygous missense mutation in PYGM segregating with the disease in the family. PYGM codes for myophosphorylase, the enzyme catalyzing the initial step of glycogen breakdown. Enzymatic tests revealed that the PYGM mutation impairs the AMP-independent myophosphorylase activity, whereas the AMP-dependent activity was preserved. Further functional investigations demonstrated an altered conformation and aggregation of mutant myophosphorylase, and the concurrent accumulation of the intermediate filament desmin in the myofibers of the patients. Interpretation: Overall, this study describes the first example of a dominant glycogen storage disease in humans, and elucidates the underlying pathomechanisms by deciphering the sequence of events from the PYGM mutation to the accumulation of glycogen in the muscle fibers. ANN NEUROL 2020;88:274–282.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as “River blindness”, a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O. volvulus ESPs to address the biology of this parasite. We showed that (1) Ov28CRP is a putative ganglioside GM2 Activator Protein (GM2AP) conserved in nematode; (2) OvGM2AP gene is transcriptionally activated in all investigated stages of the parasitic life cycle, including larval and adult stages; (3) The full-length OvGM2AP was detected in in-vitro O. volvulus ESPs of adult and larval stages; (4) the mass expressed and purified recombinant OvGM2AP purified from insect cell culture medium was found to be glycosylated at asparagine 173 and lacked N-terminal signal peptide sequence; (5) the recombinant OvGM2AP discriminated serum samples of infected and uninfected individuals; (6) OvGM2AP competitively inhibits MUG degradation by recombinant β-hexosaminidase A but not MUGS, and could not hydrolyze the GM2 to GM3; (7) humoral immune responses to the recombinant OvGM2AP revealed a negative correlation with ivermectin treatment. Altogether, our findings suggest for the first time that OvGM2AP is an antigenic molecule whose biochemical and immunological features are important to gain more insight into our understanding of host-parasite relationship, as well as its function in parasite development at large., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases. Portuguese Foundation for Science and Technology (EU/PID1003201) info:eu-repo/semantics/publishedVersion

Two distinct antagonist-bound structures of CysLT1R reveal unique ligand-binding modes and signaling mechanisms., The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs.

International audience; Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach.