FEBS Letters

doi: 10.1002/1873-3468.14396pmid: N/A

Rothman, James E.; Grushin, Kirill; Bera, Manindra; Pincet, Frederic

doi: 10.1002/1873-3468.14718pmid: 37643878

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell‐free systems suggest there are 12 SNAREpins in each ready‐release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++, and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to ‘turbocharge’ neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms hexamers that bear two copies of the v‐SNARE VAMP at each vertex, one likely assembling into a peripheral SNAREpin and the other into a central SNAREpin.

Balmas, Elisa; Sozza, Federica; Bottini, Sveva; Ratto, Maria Luisa; Savorè, Giulia; Becca, Silvia; Snijders, Kirsten Esmee; Bertero, Alessandro

doi: 10.1002/1873-3468.14709pmid: 37519013

Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.

Vignesh, Ravichandran; Aradhyam, Gopala Krishna

doi: 10.1002/1873-3468.14712pmid: 37539786

Calnuc (nucleobindin‐1, nucb1) is a Ca2+‐binding protein involved in the etiology of many human diseases. To understand the functions of calnuc, we have identified a nesfatin‐1‐like peptide (NLP) in its N terminus that is proteolyzed by a convertase enzyme in the secretory granules of cells. Mutational studies confirm the presence of a proteolytic cleavage site for proprotein convertase subtilisin/kexin type 1 (PCSK1). We demonstrate that NLP regulates Gαq‐mediated intracellular Ca2+ dynamics, likely via a G‐protein‐coupled receptor. NLP treatment to carcinoma cell lines (SCC131 cells) promotes the expression of regulators of cell cycle, proliferation, and clonogenicity by the AKT/mTOR pathway. NLP is causative of augmented migration and epithelial–mesenchymal transition (EMT), illustrating its metastatic propensity and establishing its tumor promotion ability.

Fernandes, Marie; Paget, Sonia; Kherrouche, Zoulika; Truong, Marie‐José; Vinchent, Audrey; Meneboo, Jean‐Pascal; Sebda, Shéhérazade; Werkmeister, Elisabeth; Descarpentries, Clotilde; Figeac, Martin; Cortot, Alexis B.; Tulasne, David

Umeda, Chiharu; Nakajima, Toshio; Maruhashi, Tsubasa; Tanigawa, Mirai; Maeda, Tatsuya; Mukai, Yukio

doi: 10.1002/1873-3468.14715pmid: 37574219

We previously found that overexpression of phosphate starvation‐responsive genes by disrupting PHO80 led to a shortened replicative lifespan in yeast. To identify lifespan‐related genes, we screened upregulated genes in the pho80Δ mutant and focused on the VTC genes, which encode the vacuolar polyphosphate (polyP) polymerase complex. VTC1/VTC2/VTC4 deletion restored the lifespan and intracellular polyP levels in pho80Δ. In the wild type, overexpression of VTC5 or a combination of the other VTCs caused high polyP accumulation and shortened lifespan. Similar phenotypes were caused by the deletion of polyP phosphatase genes—vacuolar PPN1 and cytosolic PPX1. The polyP‐accumulating strains exhibited stress sensitivities. Thus, we demonstrated that polyP metabolic enzymes participate in replicative lifespan, and extreme polyP accumulation shortens the lifespan.

Matsunami‐Nakamura, Risa; Tamogami, Jun; Takeguchi, Miki; Ishikawa, Junya; Kikukawa, Takashi; Kamo, Naoki; Nara, Toshifumi

doi: 10.1002/1873-3468.14711pmid: 37532685

The cell membrane of Halobacterium salinarum contains a retinal‐binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII. Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII–NpHtrII.

Wiertelak, Wojciech; Pavlovskyi, Artem; Maszczak‐Seneczko, Dorota; Szulc, Bożena; Olczak, Mariusz

doi: 10.1002/1873-3468.14714pmid: 37552213

SLC35A2 and SLC35A3 are homologous proteins with postulated nucleotide sugar transporting activities. Unlike SLC35A2, whose specificity for UDP‐Gal is well‐established, the UDP‐GlcNAc transporting activity initially attributed to SLC35A3 has recently been put into question. In this study, we constructed two hybrid proteins (SLC35A2–SLC35A3 and SLC35A3–SLC35A2) and expressed them in a previously generated SLC35A2/SLC35A3 double knockout HEK293T cell line. Our idea was to force equivalent stoichiometry of the two proteins in the cells in order to reproduce the behavior of the SLC35A2/SLC35A3 complexes in the Golgi membrane. The hybrid proteins were able to fully restore glycosylation in the double knockout. In contrast, the expression of SLC35A3 alone in these cells improved galactosylation only to a limited extent. Our study shows that the proper glycosylation requires a balanced cooperation between SLC35A2 and SLC35A3.

Mineev, Konstantin S.; Chernykh, Mikhail A.; Motov, Vladislav V.; Prudnikova, Daria A.; Pavlenko, Daniil M.; Kuzmenkov, Alexey I.; Peigneur, Steve; Tytgat, Jan; Vassilevski, Alexander A.

doi: 10.1002/1873-3468.14705pmid: 37501371

Scorpion α‐toxins (α‐NaTx) inhibiting the inactivation of voltage‐gated sodium channels (Nav) are a well‐studied family of small proteins. We previously showed that the structure of α‐NaTx specificity module responsible for selective Nav binding is governed by an interplay between the nest and niche protein motifs. Here, we report the solution structure of the toxin Lqq4 from the venom of the scorpion Leiurus quinquestriatus. Unexpectedly, we find that this toxin presents an ensemble of long‐lived structurally distinct states. We unequivocally assign these states to the alternative configurations (cis–trans isomers) of two peptide bonds: V56–P57 and C17–G18; neither of the cis isomers has been described in α‐NaTx so far. We argue that the native conformational space of α‐NaTx is wider than assumed previously.