Biology subjects

Greve, J. N.

Publications and source records attributed to Greve, J. N..

4 recordsLinked to original sources

bioRxiv2025

The SH3BGRL protein family nucleates and caps actin filaments via its conserved thioredoxin fold

Cellular actin polymerization is a tightly regulated process, typically controlled by proteins with specialized domains such as the Wiskott-Aldrich syndrome protein homology 2 (WH2) domain. Here, we identify SH3BGRL family proteins as modulators of actin dynamics, uniquely characterized by their thioredoxin (Trx) fold structure and the absence of the canonical CXXC enzymatic site essential for redox activity. The Trx fold is generally associated with enzymatic activity; however, in this context, it functions non-enzymatically to enhance actin filament nucleation and inhibit depolymerization. The family member SH3BGRL-2 was previously identified as part of the spectrin-actin complex in porcine erythrocytes. Further structural analysis reveals that human SH3BGRL proteins share structural homology with the C-terminal region of Saccharomyces cerevisiae YFR016c/Aip5, a known actin nucleation factor reported to bind G-actin. Notably, our results show that human SH3BGRL proteins do not bind G-actin directly. While they do not interact with G-actin, SH3BGRL proteins significantly increase actin assembly rates by accelerating filament nucleation without affecting barbed end elongation, as demonstrated in pyrene-actin bulk-polymerization assays and total internal reflection fluorescence microscopy (TIRFM) based single-filament studies. Furthermore, using all-atom molecular dynamics (MD) simulations and in vitro assays that directly probe the pointed end of the actin filament, we show that SH3BGRL proteins inhibit the depolymerization of existing filaments by interacting with the pointed end of the actin filament, also in the presence of the well-characterized pointed end capping protein tropomodulin. Our results indicate that all SH3BGRL family proteins promote actin nucleation by stabilizing energetically unstable actin dimers and trimers and inhibit depolymerization by direct association with the pointed end, suggesting a direct role for the Trx fold in actin dynamics.

biochemistry↗

bioRxiv2024

Classification of human actin pathological variants using C. elegans CRISPR-generated models

Actin plays a crucial role in diverse physiological processes by forming dynamic networks that determine cellular shape and mechanical properties. Non-Muscle Actinopathies (NMA) are rare diseases caused by de novo variants in human cytoskeletal {beta}-actin (ACTB) and {gamma}-actin (ACTG1) genes, ranging from missense mutations to whole gene deletions. Currently, the high clinical variability and genotype-phenotype correlations in NMA remain largely unresolved. To address this concern, we inserted nine mutations identified in patients in the C. elegans cytoplasmic actin orthologue act-2 and performed a quantitative multiscale characterization of these animal models. We uncovered various perturbations including micro-scale actin network defects, cell-scale abnormalities, morphogenesis failure, and weaker behavioural phenotypes. Notably, the severity of the observed defects correlates with the severity of patients symptoms. Thus, we provide evidence that such C. elegans models are relevant to investigate the mechanisms underlying NMA physiopathology and could ultimately be used to screen for therapeutic strategies.

cell biology↗

bioRxiv2024

Baraitser-Winter Syndrome Hotspot Mutation R196H in Cytoskeletal β-actin Reduces F-actin Stability and Perturbs Interaction with the Arp2/3 Complex

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best-defined clinical entity associated with heterozygous single-point missense mutations in cytoskeletal {beta}-actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal {beta}-actin, with the variant p.R196H being the most common. Patients carrying the p.R196H variant are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormally thick convolutions of the cerebral cortex. Here, we describe the recombinant production, purification and biochemical characterization of the BWCFF hotspot variant p.R196H. The stability and nucleotide interaction of monomeric p.R196H are unaffected, indicating a disease mechanism involving incorporation of p.R196H protomers into actin filaments. Incorporation of the variant strongly affects F-actin stability and polymerization dynamics, consistent with the position of residue R196 close to the helical axis of the actin filament and an important interstrand contact. The changes observed include an increased critical concentration of polymerization, a reduced elongation rate and an increase in the rate of filament depolymerization. In the Arp2/3-generated branch junction complex, which is essential for cell migration and endocytosis, R196 is located at the interface between the first protomer of the nucleated daughter filament and the Arp2 subunit of the Arp2/3 complex. Assays probing the interaction of p.R196H filaments with the Arp2/3 complex show a reduced efficiency of branch generation. Branch stability is impaired, as evidenced by a reduction in the number of branches and spontaneous debranching events. Furthermore, in their interaction with different types of cytoskeletal myosin motors, p.R196H filaments show isoform-specific differences. While p.R196H filaments move WT-like on lawns of surface-immobilized non-muscle myosin-2A, motility on myosin-5A is 30 % faster.

biochemistry↗

bioRxiv2023

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal gamma-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal {gamma}-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal {gamma}-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin {beta}4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q-filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q-filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin-affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover contribute to the observed disease phenotype.

biochemistry↗