Biology subjects

Zhang, T.

Publications and source records attributed to Zhang, T..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

Novel Ser74 of NF-kappaB/CgIkappaBalpha Phosphorylated by MAPK/ERK Regulates Temperature Adaptation in Oysters

Phosphorylation of I{kappa}B at Ser32 and Ser36 by IKKs during biotic stress triggers its ubiquitin-proteasome degradation, causing to the nuclear translocation of REL, representing a key cascade mechanism in metazoans conserved and immune core signaling pathway, NF-{kappa}B. However, studies on its response to abiotic stress and signal transduction by phosphorylation in mollusks are lacking. Here, we firstly report a novel heat-induced phosphorylation site (Ser74) at the major NF-{kappa}B/CgI{kappa}B of oysters, phosphorylated by MAPK/CgERK1/2, which independently mediated the subsequent ubiquitin-proteasome degradation without phosphorylation at Ser32 and Ser36 and decreased thermal stability. The degradation of CgI{kappa}B promoted CgREL nuclear translocation, which stimulated cell survival related gene expression to defend against thermal stress. The MAPK and NF-{kappa}B pathways exhibited stronger activation patterns in higher environmental temperature and in the warm-adapted Crassostrea angulata than those in the cold-adapted C. gigas-two allopatric congeneric oyster species with differential habitat temperatures. These findings unveil the complex and unique phosphorylation-mediated signal transduction mechanisms in marine invertebrates, and further expand our understanding of the evolution and function of established classical pathway crosstalk mechanisms.

cell biology↗

bioRxiv2024

High-throughput Screening of Compounds against Autoluminescent Nonreplicating Mycobacterium tuberculosis under Diverse Conditions

The screening of new anti-mycobacterial chemicals is primarily focused on inhibiting the active growing bacteria. However, a major challenge in tuberculosis control is the ability of Mycobacterium tuberculosis to enter a nonreplicating state for extended periods, rendering it resistant to many clinical drugs and complicating eradication efforts. Existing low-oxygen-recovery assays designed for screening compounds targeting nonreplicating M. tuberculosis have limitations, including the colony-forming unit counting for non-luminous M. tuberculosis and the instability of the free plasmid carrying luxAB genes in luminescent M. tuberculosis, along with exogenous substrate requirements for light producing. Moreover, these assays fail to accurately replicate the growth conditions of nonreplicating M. tuberculosis in vitro, thus resulting in less convincing results. To address these challenges, we have developed an autoluminescence-based, cholesterol-enriched culture evaluation model to assess 17 anti-tuberculosis drugs of different classes against nonreplicating M. tuberculosis. Our findings indicate that the relative light unit, measured in real-time, serves as a reliable surrogate marker for colony-forming unit, which typically becomes available one month later. This suggests the utility of our model for the rapid determination of drug susceptibility dynamically. The autoluminescent M. tuberculosis, harbouring luxCDABE gene cluster within its genome, can emit blue-green light stably and autonomously without requiring an external substrate supplement. The minimal inhibitory concentrations of all the drugs tested under anaerobic conditions are significantly different from that detected in aerobic environment. Our model allows for rapid, precise, and efficient assessment of drug activity under anaerobic conditions, thereby enabling a more comprehensive evaluation of anti-mycobacterial efficacy. Overall, our model represents a significant advancement in anti-tuberculosis drug discovery and pharmaceutical development.

microbiology↗

bioRxiv2024

The catabolism of alanine and glutamate ensure proper sporulation by respectively preventing premature germination and providing energy sources

Sporulation as a typical bacterial differentiation process has been studied for decades. However, two crucial aspects of sporulation, (i) the energy sources supporting the process, and (ii) the maintenance of spore dormancy throughout sporulation, are scarcely explored. Here, we reported the crucial role of RocG-mediated glutamate catabolism in regulating mother cell lysis, a critical step for successful sporulation, likely by providing energy metabolite ATP. Notably, rocG overexpression resulted in an excessive ATP accumulation in sporulating cells, leading to adverse effects on future spore properties, e.g. increased germination efficiency, reduced DPA content, and lowered heat resistance. Additionally, we revealed that Ald-mediated alanine metabolism decreased the typical germinant L-alanine concentration in sporulating environment, thereby preventing premature germination and maintaining spore dormancy. Our data inferred that sporulation was a highly orchestrated biological process requiring a delicate balance in diverse metabolic pathways, hence ensuring both the completion of sporulation and production of high-quality spores.

microbiology↗

bioRxiv2024

Conserved helical motifs in the Ikaros IDR mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1/Ikaros is essential for B cell development, and recurrently mutated in human B-ALL. Ikaros has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of Ikaros-associated coregulatory complexes and their contribution to Ikaros-mediated gene regulation are not well understood. To address this issue, we performed an unbiased identification of Ikaros-interacting proteins in pre-B cells, and found that Ikaros interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to Ikaros induction. Transcriptional repression preceded transcriptional activation by several hours, and was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. Functional characterisation of intrinsically disordered regions in the Ikaros protein identified highly conserved helical motifs that mediate Ikaros association with the NuRD corepressor complex and contribute to the silencing of target genes in pre-B cells and antiproliferative functions of Ikaros in human B-ALL.

molecular biology↗

bioRxiv2024

High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants

Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools to install precise C-to-T and A-to-G base edits in living organisms. In recently years, a highly efficient ABE based on engineered TadA-8e, known as ABE8e, has been developed to confer highly efficient A-to-G editing in mammalian cells and plants. Interestingly, TadA-8e can be further evolved and engineered to confer cytosine base editing. In this study, we compare in rice and tomato cells a serial of engineered CBEs derived from TadA-8e and identify TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high editing purity, while maintaining the relatively narrow editing window of ABE8e. Excitingly, we show TadDE is a new class of dual base editor where a single deaminase domain promotes simultaneous C-to-T and A-to-G base editing in plant cells. Multiplexed base editing is demonstrated in transgenic rice plants with TadCBEa and TadDE. Furthermore, no off-target effects of TadCBEa and TadDE can be detected by whole genome and transcriptome sequencing of the transgenic lines, suggesting high editing specificity. Finally, we demonstrate two gain-of-function crop engineering applications in rice by using the efficient dual base editor, TadDE. In the first case, herbicide resistance alleles of OsALS are introduced by TadDE. In the second case, synonymous mutations are generated in OsSPL14, making its transcript resistant to OsMIR156-mediated degradation. Together, this study reports TadA-8e derived CBEs and a dual base editor with high editing activity, purity, and specificity. They are valuable additions to the expanding plant base editing toolbox.

molecular biology↗

bioRxiv2024

Ilamycins are dual inhibitors of ClpX and ClpC1 involved in proteostasis in Mycobacteria

The mycobacterial caseinolytic protease (Clp) system has been recognized as a promising therapeutic target. In this study, we identified two novel ilamycin analogs, ilamycin E (ILE) and ilamycin F (ILF), both targeting the ClpC1 component of the ClpC1P1P2 proteasome. ILE potently disrupts ClpC1P1P2-mediated proteolysis, leading to delayed bactericidal activity, while ILF also binds ClpC1, albeit with lower affinity. Notably, we discovered a unique mutation in clpX and a novel insertion in clpC1, both conferring resistance to ILE and ILF in mycobacterium, validated by gene editing. Furthermore, ILE can also inhibit the proteolytic activity of ClpXP1P2 in a manner dependent on the substrates tag sequence and adaptor. This first demonstration of clpX- and clpC1-mediated ilamycin resistance underscores the potential of ilamycins to target multiple components of the Clp protease system, offering a novel dual-target strategy for combating mycobacterial infections.

microbiology↗

bioRxiv2024

EmbB and EmbC Regulate the Sensitivity of Mycobacterium abscessus to Echinomycin

Treatment of Mycobacterium abscessus (Mab) infection is a major challenge due to its intrinsic resistance to most available drugs. It is thus imperative to find new anti-Mab drugs. In this study, we investigated the activity and intrinsic resistance mechanism of echinomycin (ECH) against Mab. ECH is active against Mab (MIC: 2 {micro}g/mL). The embC gene knockout strain (Mab{Delta}embC) showed hyper-sensitive to ECH (MIC: 0.0078-0.0156 {micro}g/mL). The MICs of ECH-resistant strains screened based on the Mab{Delta}embC strain were 0.25-1 {micro}g/mL. Mutations were found in the EmbB, including Asp306Ala, Asp306Asn, Arg350Gly, Val555Ile, and Gly581Ser, which led to increased resistance to ECH when overexpressed in Mab{Delta}embC individually (0.25-0.5 {micro}g/mL). The EmbB mutants edited by the CRISPR/Cpf1 system became more resistant to ECH (MIC: 0.25-0.5 {micro}g/mL). The permeability of gene-edited and overexpressed Mab strains was reduced, as shown by the ethidium bromide accumulation assay, but it was still significantly higher than that of the parent Mab. To summarize, our study demonstrates that ECH has a strong anti-Mab activity and confirms that EmbB and EmbC are related to the sensitivity of Mab to ECH. EmbB mutation may partially compensate for the function of EmbC. Impact StatementMycobacterium abscessus (Mab) is a rapidly growing, intrinsic multidrug-resistant Mycobacterium. This study demonstrated that echinomycin (ECH) has potent antibacterial activity against Mab, and the mechanism of ECH resistance to Mab is related to EmbB and EmbC. EmbB and EmbC can alter the sensitivity of Mab to ECH by altering the permeability of its cell wall. In addition, there is a functional complementary evolution between EmbB and EmbC to regulate sensitivity to ECH. Overall, our study provides a novel anti-Mab drug candidate ECH and a scientific foundation for developing effective strategies to prevent and control Mab.

microbiology↗

bioRxiv2024

Ubiquitin-mediated stabilization of SlPsbS regulates low night temperature tolerance in tomatoes

Non-photochemical quenching (NPQ) plays a fundamental role in safely operating photosynthesis under low night temperatures (LNT). PsbS protein is essential for the rapid induction of NPQ, and its stability is often affected by adverse environmental conditions. However, the regulatory mechanism for the stability of PsbS or chloroplast proteins remains to be fully characterized. We showed that LNT decreased NPQ levels and SlPsbS protein abundance in tomato leaves. COP9 signalosome subunit 5A (SlCSN5A) facilitated SlPsbS ubiquitination and degradation in the cytosol. Further, tomato chloroplast vesiculation (SlCV) was activated by LNT. Under LNT, SlCV targeted the chloroplasts and induced the formation of CV-containing vesicles (CCVs) containing SlPsbS, which were exported from the chloroplasts. Subsequently, SlCV and SlPsbS contact SlCSN5A in the cytosol and are ubiquitinated and degraded. Genetic evidence demonstrated that overexpression of SlCV aggravated SlPsbS protein degradation, whereas silencing of SlCSN5 and SlCV delayed LNT-induced NPQ reduction and SlPsbS protein turnover. This study provides evidence that CSN5A is associated with chloroplast proteins, and reveals a ubiquitin-dependent degradation pathway of chloroplast proteins co-mediated by CV and CSN5A, thereby providing new insights into the regulation of chloroplast protein stability under stress conditions.

plant biology↗

bioRxiv2024

The first discovery of SFTSV in the Centre of Metropolitan Beijing, China

Severe fever with thrombocytopenia virus (SFTSV), an emerging tick-borne bandavirus, poses a significant public health threat in rural China. Since 2021, an increase in local cases has been noted in the rural-urban fringe surrounding Beijing. This study aimed to assess the formation of natural foci in urban areas by conducting a field survey of ticks and hedgehogs from Beijings second to fifth ring roads. Our survey revealed a diverse tick population in city parks, including the major SFTSV vector, H. longicornis. Parthenogenetic H. longicornis, known for its role in rapid SFTSV spread, was identified in key locations like Beihai Park and Taoranting Park, near the Forbidden City. Notably, high SFTSV seroprevalence and RNA prevalence were found in hedgehogs and parasitic ticks in the center of Beijing. These findings highlight the circulation of SFTSV in central Beijing, underscoring the need for urgent attention and enhanced surveillance measures.

microbiology↗

bioRxiv2024

Hosts Manipulate Lifestyle Switch and Pathogenicity Heterogeneity of Opportunistic Pathogens in the Single-cell Resolution

Host-microbe interactions are virtually bidirectional, but how the host affects their microbiome is poorly understood. Here, we report that the host is a critical modulator to regulate the lifestyle switch and pathogenicity heterogeneity of the opportunistic pathogens Serratia marcescens utilizing the Drosophila and bacterium model system. First, we find that Drosophila larvae efficiently outcompete S. marcescens and typically drive a bacterial switch from pathogenicity to commensalism toward the fly. Furthermore, Drosophila larvae reshape the transcriptomic and metabolic profiles of S. marcescens characterized by a lifestyle switch. More important, the host alters pathogenicity and heterogeneity of S. marcescens in the single-cell resolution. Finally, we find that larvae-derived AMPs are required to recapitulate the response of S. marcescens to larvae. Altogether, our findings provide an insight into the pivotal roles of the host in harnessing the life history and heterogeneity of symbiotic bacterial cells, advancing knowledge of the reciprocal relationships between the host and pathogen.

microbiology↗

bioRxiv2024

Reducing stomatal density by expression of a synthetic EPF increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Reducing stomatal conductance (gs) through manipulating stomatal density has been observed to translate to improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to impair AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, lead to reduced stomatal densities. A moderate reduction in stomatal density did not strengthen stomatal limitation to AN, improved WUE, reduced water use, and avoided loss of carbon fixation during a period of water deprivation. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues provides a potential pathway to optimal agronomic outcomes.

plant biology↗

bioRxiv2024

reguloGPT: Harnessing GPT for Knowledge Graph Construction of Molecular Regulatory Pathways

MotivationMolecular Regulatory Pathways (MRPs) are crucial for understanding biological functions. Knowledge Graphs (KGs) have become vital in organizing and analyzing MRPs, providing structured representations of complex biological interactions. Current tools for mining KGs from biomedical literature are inadequate in capturing complex, hierarchical relationships and contextual information about MRPs. Large Language Models (LLMs) like GPT-4 offer a promising solution, with advanced capabilities to decipher the intricate nuances of language. However, their potential for end-to-end KG construction, particularly for MRPs, remains largely unexplored. ResultsWe present reguloGPT, a novel GPT-4 based in-context learning prompt, designed for the end-to-end joint name entity recognition, N-ary relationship extraction, and context predictions from a sentence that describes regulatory interactions with MRPs. Our reguloGPT approach introduces a context-aware relational graph that effectively embodies the hierarchical structure of MRPs and resolves semantic inconsistencies by embedding context directly within relational edges. We created a benchmark dataset including 400 annotated PubMed titles on N6-methyladenosine (m6A) regulations. Rigorous evaluation of reguloGPT on the benchmark dataset demonstrated marked improvement over existing algorithms. We further developed a novel G-Eval scheme, leveraging GPT-4 for annotation-free performance evaluation and demonstrated its agreement with traditional annotation-based evaluations. Utilizing reguloGPT predictions on m6A-related titles, we constructed the m6A-KG and demonstrated its utility in elucidating m6As regulatory mechanisms in cancer phenotypes across various cancers. These results underscore reguloGPTs transformative potential for extracting biological knowledge from the literature. Availability and implementationThe source code of reguloGPT, the m6A title and benchmark datasets, and m6A-KG are available at: https://github.com/Huang-AI4Medicine-Lab/reguloGPT.

bioinformatics↗

bioRxiv2024

Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in Mycobacterium abscessus

Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes many types of serious infections in humans, posing a significant public health challenge. Currently, effective genetic manipulation tools for M. abscessus are still being developed, which hampers research and therapeutic development. However, the clustered regularly interspaced short palindromic repeats (CRISPR) - associated protein (Cas) systems have emerged as promising methods for generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy in M. abscessus. Additionally, we discovered that NrgA from Mycobacterium marinum is crucial for the repair of DSBs caused by the CRISPR-Cas system in M. abscessus. Finally, contrary to previous findings, our study also indicates that inhibiting or overexpressing homologous recombination/single-strand annealing significantly decreases the efficiency of NHEJ repair in M. abscessus. This discovery challenges established perspectives and suggests that the NHEJ repair in M. abscessus may require the involvement of components from homologous recombination and single-strand annealing, demonstrating the complex interactions among the three DSB repair pathways in M. abscessus. Impact statementThere are still very few genetic manipulation tools available for Mycobacterium abscessus. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing in M. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption or overexpression of either HR or single-strand annealing not only failed to enhance but also significantly reduced the gene editing efficiency in M. abscessus. This suggests that NHEJ repair in M. abscessus may require components from both HR and single-strand annealing, highlighting a complex interaction among the DSB repair pathways in M. abscessus.

microbiology↗

bioRxiv2024

Early evolution of the ecdysozoan body plan

Extant ecdysozoans (moulting animals) are represented by a great variety of vermiform or articulated organisms. However, controversies remain about the nature of their ancestral body plan although the vermiform hypothesis seems to prevail. We describe here Beretella spinosa gen et sp. nov. a tiny ecdysozoan from the early Cambrian, Yanjiahe Formation, South China, with an unusual sack-like appearance, single opening, and spiny ornament. Beretella has no equivalent among animals, except Saccorhytus from the basal Cambrian. Phylogenetic analyses resolve both forms as a sister group (Saccorhytida) to all known Ecdysozoa, thus suggesting that ancestral ecdysozoans may have been non-vermiform animals. Saccorhytids are likely to represent an early dead-end off-shot along the stem-line Ecdysozoa that possibly evolved through anatomical simplification (e.g. lack of anus). Although extinct during the Cambrian, this animal lineage provides precious insight into the early evolution of Ecdysozoa and the nature (possibly non-vermiform) of the earliest representatives of the group.

paleontology↗

bioRxiv2023

Pharmacokinetics, Tissue Distribution, and Formulation Study of a Small-molecule Inhibitor of MKLP2, LG157

LG157 is a recently identified small-molecule inhibitor of mitotic kinesin-like protein 2 (MKLP2), an overlooked oncology target. This study aims to explore the drug developability of LG157, by assessing its druglike properties, determining plasma drug exposure in various oral formulations, and exploring the self-emulsifying drug delivery system (SEDDS). Solubility of LG157 ranges from 175 to 228 M across pH 1.0 to 13.0, with a LogD of 2.41 at pH 7.4. It showed a high protein binding rate of 92.58% in mouse plasma and 90.30% in human serum. The bioavailability radar plot aligns with experimental data (69-85%), indicating good bioavailability. In line with the computation prediction, preclinical formulation studies in mice reveal that all five formulations tested offer decent plasma LG157 exposure, with the highest level of LG157 exposure in the PEG300-based formulation. Subsequent tissue distribution studies in rats indicated that the compound is widely distributed with the highest concentration of LG157 in the liver and the lowest level in the brain. The optimal SEDDS formulation, SEDDS-F14, consists of 65% Oleic acid, 26.25% Tween 20, and 8.75% PEG400 as oil, surfactant, and co-surfactant, respectively. SEDDS optimization, based on the central composite design, has achieved the maximum loading of 188.7 mg/mL for LG157. These findings support the developability of LG157 and encourage continued exploration and refinement of formulations for improved therapeutic efficacy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/573327v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@11b4bb7org.highwire.dtl.DTLVardef@107e320org.highwire.dtl.DTLVardef@d06f55org.highwire.dtl.DTLVardef@3d4854_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Brg1 regulates fibroblast-myofibroblast transition to promote renal fibrosis

Excessive fibrogenesis serves to disrupt the anatomical and functional integrity of the kidneys contributing to renal failure. Renal fibroblast is the major precursor to myofibroblast, the effector cell type of renal fibrosis. How fibroblast-myofibroblast transition (FMyT) is regulated in the kidneys remains incompletely understood. In the present study we investigated the role of Brahma related gene 1 (Brg1), a chromatin remodeling protein, in renal fibrosis focusing on mechanistic insights and translational potential. We report that Brg1 was up-regulated during FMyT both in vitro and in vivo. Brg1 deletion in fibroblasts partially blocked TGF-{beta} induced FMyT in vitro and attenuated renal fibrosis in three different animal models. Importantly, conditional Brg1 knockout in Postn+ mature myofibroblasts mitigated renal fibrosis induced by unilateral ureteral obstruction (UUO) or ischemia-reperfusion (IR) in mice. Transcriptomic analysis uncovered Prune2 as a potential target for Brg1. Brg1 interacted with E2F1 to activate Prune2 transcription during FMyT. Concordantly, Prune2 knockdown suppressed TGF-{beta} induced FMyT in vitro and dampened renal fibrosis in mice. Mechanistically, Prune2 likely contributed to FMyT by augmenting phosphorylation and activity of the pro-fibrogenic transcription factor PU.1. Finally, small-molecule Brg1 inhibitor PFI-3 exhibited strong antifibrotic potency in established models of renal fibrosis. In conclusion, our data provide compelling evidence that BRG1 is a pivotal regulator of as well as a promising therapeutic target for renal fibrosis.

pharmacology and toxicology↗

bioRxiv2023

FGF12 Modulates Cardiomyocyte Hypertrophy by Inhibiting GATA4-ERK1/2 Phosphorylation Through Altered Intracellular Localization

BackgroundHypertrophic cardiomyopathy (HCM) is a heritable pathological condition resulting from mutations in sarcomere-related proteins, leading to severe structural abnormalities without effective treatment options. Although reduced fibroblast growth factor 12(FGF12) expression is observed in HCM patients, its functional role remains unclear. MethodsEmploying immunoprecipitation (IP)-mass spectrometry (MS) and CUT&Tag sequencing, we investigated FGF12-interacting proteins in myocardial samples from healthy volunteers and HCM patients. CRISPR-Cas9 was utilized to explore the function and interaction partners of FGF12 in cardiomyocytes induced from human pluripotent stem cells (hiPSCs-CMs), other cell lines, and mouse models (MYH7R403Q and MYBPC3c.790G>A, transverse aortic constriction (TAC)). During hypertrophy, FGF12 localizes intranuclearly, prompting investigations into its binding to gene promoter regions through CUT&Tag sequencing and dual-luciferase experiments using myocardial tissues from patients. The beating frequency of hiPS-CMs was assessed using the CardioExcyte 96 real-time label-free cardiomyocyte functional analysis system. ResultsFGF12 was found to associate with proteins involved in energy metabolism, predominantly localizing to the perinuclear space under physiological conditions but shifting into the nucleus of hypertrophic cardiomyocytes. Co-IP-MS revealed significant interactions between FGF12 and metabolism-associated proteins, particularly GATA binding protein 4 (GATA4) and mitogen-activated protein kinase 1/3 (MAPK1/3) in the perinuclear space. In a hypertrophic state, FGF12 bound to the GATA4 promoter region, increasing its expression upon nuclear translocation. Both in vitro and in vivo models demonstrated that FGF12 interaction with GATA4 inhibited GATA4 and MAPK1/3 phosphorylation, inducing the expression of hypertrophy-associated genes. Overexpression of FGF12-NLS-del (nuclear localization signal deletion) resulted in decreased GATA4 phosphorylation, suggesting inhibition in the perinuclear region. ConclusionsThis study elucidates a pathological mechanism of HCM involving FGF12, where its nuclear localization enhances phosphorylation, GATA4 expression, and activation of the ERK1/2-pGATA4 pathway genes associated with hypertrophy. Beyond advancing our understanding of HCM, these findings propose FGF12 as a potential therapeutic target for HCM, warranting further exploration to potentially alleviate this condition affecting millions of individuals.

molecular biology↗

bioRxiv2023

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 [A] resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

cell biology↗