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The Journal of Neuroscience : the... Jul 2024The release of neurotransmitters at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Amongst those dedicated...
The release of neurotransmitters at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Amongst those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind SNARE complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin II (Cpx). The N-terminus (amino acid 1 - 27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release, but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine tunes the degree of spontaneous and evoked neurotransmitter release. We describe in this work the importance of the N-terminal domain of the small regulatory cytosolic protein complexin in spontaneous and evoked glutamatergic neurotransmitter release at hippocampal mouse neurons. We use biochemical assays to screen for amino acids of interest in the complexin N-terminus and test these residues for functional relevance in spontaneous and Ca-triggered synaptic vesicle exocytosis using electrophysiology assays and site-directed mutagenesis. In addition to identifying crucial residues for clamping spontaneous release and promoting Ca-evoked transmission, we identify a single amino acid at position D15 which determines synaptic vesicle priming, a function that was never before attributed to complexin at vertebrate synapses.
PubMed: 38951039
DOI: 10.1523/JNEUROSCI.0076-24.2024 -
The Journal of Neuroscience : the... Jul 2024At chemical synapses, voltage-gated Ca-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca microdomains they...
At chemical synapses, voltage-gated Ca-channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca microdomains they elicit must be located precisely to primed SVs, to evoke rapid transmitter release. Localization is mediated by Rab3 interacting molecule (RIM) and RIM-binding proteins (RIM-BPs), which interact and bind to the C-terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction (NMJ) of hermaphrodites. mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Even though postsynaptic depolarization was reduced, mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABA or GABA receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus RIMB-1 function differentially affects excitation/inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission in mixed circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the defects, and similar phenotypes resulted from acute degradation of the CaV2 β-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerges from VGCC regulation. The machinery for chemical synaptic transmission is organized in a precise spatial arrangement in order to enable efficient and temporally accurate coupling of action potentials with the rise of the Ca concentration through CaV2 P/Q-type voltage gated Ca channels. This triggers the fusion of synaptic vesicles with the plasma membrane and the release of transmitters. Here, we analyzed the molecular and functional interplay of proteins of the active zone scaffold, RIM and RIM-binding protein (RIMB-1), with the CaV2 channel in the neuromuscular junction, a tripartite synapse with cholinergic and GABAergic neuronal input. Our work shows a differential requirement of RIMB-1 in cholinergic vs. GABAergic neurons, that affects the regulation of excitation-inhibition balance at circuit, cellular and ultrastructural levels.
PubMed: 38951038
DOI: 10.1523/JNEUROSCI.0535-22.2024 -
Nucleic Acids Research Jul 2024In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic...
In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.
PubMed: 38950903
DOI: 10.1093/nar/gkae520 -
The Journal of Clinical Investigation Jun 2024Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML),...
Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.
Topics: Humans; Leukemia, Myeloid, Acute; fms-Like Tyrosine Kinase 3; Drug Resistance, Neoplasm; Animals; Mice; Niacinamide; Cell Line, Tumor; Xenograft Model Antitumor Assays; Female; Antineoplastic Agents; Mutation; Mice, SCID; Mice, Inbred NOD
PubMed: 38950330
DOI: 10.1172/JCI169245 -
PLoS Biology Jul 2024We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis...
We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis development, recombination initiation, or recombination-mediated homolog coalignment/pairing. In striking contrast, Hop1 plays a critical role at the leptotene/zygotene transition which is defined by transition from pairing to synaptonemal complex (SC) formation. During this transition, Hop1 is required for maintenance of normal axis structure, formation of SC from telomere to telomere, and development of recombination foci. These hop1Δ mutant defects are DSB dependent and require Sme4/Zip1-mediated progression of the interhomolog interaction program, potentially via a pre-SC role. The same phenotype occurs not only in hop1Δ but also in absence of the cohesin Rec8 and in spo76-1, a non-null mutant of cohesin-associated Spo76/Pds5. Thus, Hop1 and cohesins collaborate at this crucial step of meiotic prophase. In addition, analysis of 4 non-null mutants that lack this transition defect reveals that Hop1 also plays important roles in modulation of axis length, homolog-axis juxtaposition, interlock resolution, and spreading of the crossover interference signal. Finally, unexpected variations in crossover density point to the existence of effects that both enhance and limit crossover formation. Links to previously described roles of the protein in other organisms are discussed.
PubMed: 38950075
DOI: 10.1371/journal.pbio.3002705 -
Cancer Research Communications Jul 2024Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by...
Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases. Myristoylation is emerging as a promising cancer therapeutic target, however the molecular determinants of sensitivity to N-myristoyltransferase inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that N-myristoyltransferases are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter Translocase of Inner Mitochondrial Membrane 17 homologue A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis N-myristoyltransferase-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.
PubMed: 38949950
DOI: 10.1158/2767-9764.CRC-23-0428 -
The Plant Journal : For Cell and... Jul 2024Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle...
Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-B*) was inhibited early during initial penetration, with the TaICL-B* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.
PubMed: 38949911
DOI: 10.1111/tpj.16908 -
International Journal of Cancer Jul 2024Gliomas are primary brain tumors and are among the most malignant types. Adult-type diffuse gliomas can be classified based on their histological and molecular...
Gliomas are primary brain tumors and are among the most malignant types. Adult-type diffuse gliomas can be classified based on their histological and molecular signatures as IDH-wildtype glioblastoma, IDH-mutant astrocytoma, and IDH-mutant and 1p/19q-codeleted oligodendroglioma. Recent studies have shown that each subtype of glioma has its own specific distribution pattern. However, the mechanisms underlying the specific distributions of glioma subtypes are not entirely clear despite partial explanations such as cell origin. To investigate the impact of multi-scale brain attributes on glioma distribution, we constructed cumulative frequency maps for diffuse glioma subtypes based on T1w structural images and evaluated the spatial correlation between tumor frequency and diverse brain attributes, including postmortem gene expression, functional connectivity metrics, cerebral perfusion, glucose metabolism, and neurotransmitter signaling. Regression models were constructed to evaluate the contribution of these factors to the anatomic distribution of different glioma subtypes. Our findings revealed that the three different subtypes of gliomas had distinct distribution patterns, showing spatial preferences toward different brain environmental attributes. Glioblastomas were especially likely to occur in regions enriched with synapse-related pathways and diverse neurotransmitter receptors. Astrocytomas and oligodendrogliomas preferentially occurred in areas enriched with genes associated with neutrophil-mediated immune responses. The functional network characteristics and neurotransmitter distribution also contributed to oligodendroglioma distribution. Our results suggest that different brain transcriptomic, neurotransmitter, and connectomic attributes are the factors that determine the specific distributions of glioma subtypes. These findings highlight the importance of bridging diverse scales of biological organization when studying neurological dysfunction.
PubMed: 38949756
DOI: 10.1002/ijc.35068 -
ELife Jul 2024Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation;...
Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.
Topics: Chemokine CCL5; Animals; Heparitin Sulfate; Humans; Cricetulus; CHO Cells; Mice; Chemotaxis; Heparin; Phase Separation
PubMed: 38949655
DOI: 10.7554/eLife.93871 -
ELife Jul 2024Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of...
Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of . We found that the status of β-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.
Topics: Animals; Tubulin; Protein Processing, Post-Translational; Caenorhabditis elegans; Microtubules; Caenorhabditis elegans Proteins; Acetylation; Axons; Phosphorylation; Nerve Regeneration; Kinesins
PubMed: 38949652
DOI: 10.7554/eLife.94583