Protein Subcellular Localization Research Articles

Bimolecular Fluorescence Complementation (BiFC) in Host-Virus Interactions.

Bimolecular fluorescence complementation (BiFC) is an assay widely used for studying protein-protein interactions and determining the subcellular localization of proteins. This technique involves fusing the proteins of interest to separate structural domains of a fluorescent protein, followed by transient expression in cells. The interaction between the proteins of interest in vivo allows the reconstitution of the fluorescence that can be visualized by fluorescence microscopy. BiFC has been particularly useful in investigating the interactions between viral and host proteins. Here, we describe the steps involved in preparing expression cassettes that allow the expression of proteins of interest fused to nonfluorescent fragments of yellow fluorescent protein (YFP), Agrobacterium transformations, and agroinfiltration of Nicotiana benthamiana leaves to facilitate virus protein-host protein interactions. Finally, high-resolution images can be obtained by analyzing the leaves under a confocal microscope.

Essential proteins discovery based on dominance relationship and neighborhood similarity centrality.

Essential proteins play a vital role in development and reproduction of cells. The identification of essential proteins helps to understand the basic survival of cells. Due to time-consuming, costly and inefficient with biological experimental methods for discovering essential proteins, computational methods have gained increasing attention. In the initial stage, essential proteins are mainly identified by the centralities based on protein-protein interaction (PPI) networks, which limit their identification rate due to many false positives in PPI networks. In this study, a purified PPI network is firstly introduced to reduce the impact of false positives in the PPI network. Secondly, by analyzing the similarity relationship between a protein and its neighbors in the PPI network, a new centrality called neighborhood similarity centrality (NSC) is proposed. Thirdly, based on the subcellular localization and orthologous data, the protein subcellular localization score and ortholog score are calculated, respectively. Fourthly, by analyzing a large number of methods based on multi-feature fusion, it is found that there is a special relationship among features, which is called dominance relationship, then, a novel model based on dominance relationship is proposed. Finally, NSC, subcellular localization score, and ortholog score are fused by the dominance relationship model, and a new method called NSO is proposed. In order to verify the performance of NSO, the seven representative methods (ION, NCCO, E_POC, SON, JDC, PeC, WDC) are compared on yeast datasets. The experimental results show that the NSO method has higher identification rate than other methods.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis

BackgroundUDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides.ResultsIn this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark.ConclusionOur findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Dual-Signal Feature Spaces Map Protein Subcellular Locations Based on Immunohistochemistry Image and Protein Sequence.

Protein is one of the primary biochemical macromolecular regulators in the compartmental cellular structure, and the subcellular locations of proteins can therefore provide information on the function of subcellular structures and physiological environments. Recently, data-driven systems have been developed to predict the subcellular location of proteins based on protein sequence, immunohistochemistry (IHC) images, or immunofluorescence (IF) images. However, the research on the fusion of multiple protein signals has received little attention. In this study, we developed a dual-signal computational protocol by incorporating IHC images into protein sequences to learn protein subcellular localization. Three major steps can be summarized as follows in this protocol: first, a benchmark database that includes 281 proteins sorted out from 4722 proteins of the Human Protein Atlas (HPA) and Swiss-Prot database, which is involved in the endoplasmic reticulum (ER), Golgi apparatus, cytosol, and nucleoplasm; second, discriminative feature operators were first employed to quantitate protein image-sequence samples that include IHC images and protein sequence; finally, the feature subspace of different protein signals is absorbed to construct multiple sub-classifiers via dimensionality reduction and binary relevance (BR), and multiple confidence derived from multiple sub-classifiers is adopted to decide subcellular location by the centralized voting mechanism at the decision layer. The experimental results indicated that the dual-signal model embedded IHC images and protein sequences outperformed the single-signal models with accuracy, precision, and recall of 75.41%, 80.38%, and 74.38%, respectively. It is enlightening for further research on protein subcellular location prediction under multi-signal fusion of protein.

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM

Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function.

Identification and characteristics of temperature-sensitive sites of Pseudomonas plecoglossicida based on aptamers

Pseudomonas plecoglossicida can infect Pseudosciaena crocea and cause visceral white spot disease, resulting in substantial economic losses to the P. crocea industry. Pseudomonas plecoglossicida is a temperate-sensitive bacterium and only infects P. crocea below 22 ℃. Aptamers are oligonucleotide molecules selected by the systematic evolution of ligands by exponential enrichment (SELEX) and can identify all kinds of targets. In the present study, five aptamers (M17, M19, M48, M56 and M81) for P. plecoglossicida cultured at 18 ℃ were selected by SELEX and based on comparative analysis of the high frequency sequences. The five aptamers had significantly higher affinities for the pathogenic P. plecoglossicida at 18 ℃ than for the nonpathogenic P. plecoglossicida at 28 ℃ and the other six bacteria (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Vibiro harveryi, Vibiro alginolyticus, Pseudomonas aeruginosa) (P < 0.01). The affinity constants (Kd) and maximum affinity (Am) of these aptamers for P. plecoglossicida were determined, with Kd values of M17 (23.35 ± 6.40), M19 (66.37 ± 14.73), M48 (105.91 ± 21.41), M56 (31.16 ± 5.03) and M81 (52.63 ± 13.82) nmol/L and with Am values of M17 (1041.26 ± 57.08), M19 (2291.76 ± 171.96), M48 (1258.51 ± 21.41), M56 (1307.70 ± 54.01) and M81 (543.27 ± 44.16) nmol/L, respectively. The secondary structures of the aptamers were analyzed, and the aptamer binding proteins of the pathogenic P. plecoglossicida were isolated by magnetic separation and purified by polyacrylamide gel electrophoresis. Mass spectrometry revealed ribosomal proteins to be the main aptamer binding proteins. The secondary and tertiary structures and subcellular localization of these aptamer binding proteins were also studied and analyzed. These findings provide a foundation for the temperature-sensitive pathogenic mechanism of P. plecoglossicida in P. crocea. This is the first report on aptamers that recognize temperature-sensitive sites in P. plecoglossicida.

RAB1A haploinsufficiency phenocopies the 2p14–p15 microdeletion and is associated with impaired neuronal differentiation

Hereditary spastic parapareses (HSPs) are clinically heterogeneous motor neuron diseases with variable age of onset and severity. Although variants in dozens of genes are implicated in HSPs, much of the genetic basis for pediatric-onset HSP remains unexplained. Here, we re-analyzed clinical exome-sequencing data from siblings with HSP of unknown genetic etiology and identified an inherited nonsense mutation (c.523C>T [p.Arg175Ter]) in the highly conserved RAB1A. The mutation is predicted to produce a truncated protein with an intact RAB GTPase domain but without two C-terminal cysteine residues required for proper subcellular protein localization. Additional RAB1A mutations, including two frameshift mutations and a mosaic missense mutation (c.83T>C [p.Leu28Pro]), were identified in three individuals with similar neurodevelopmental presentations. In rescue experiments, production of the full-length, but not the truncated, RAB1a rescued Golgi structure and cell proliferation in Rab1-depleted cells. In contrast, the missense-variant RAB1a disrupted Golgi structure despite intact Rab1 expression, suggesting a dominant-negative function of the mosaic missense mutation. Knock-down of RAB1A in cultured human embryonic stem cell-derived neurons resulted in impaired neuronal arborization. Finally, RAB1A is located within the 2p14-p15 microdeletion syndrome locus. The similar clinical presentations of individuals with RAB1A loss-of-function mutations and the 2p14-p15 microdeletion syndrome implicate loss of RAB1A in the pathogenesis of neurodevelopmental manifestations of this microdeletion syndrome. Our study identifies a RAB1A-related neurocognitive disorder with speech and motor delay, demonstrates an essential role for RAB1a in neuronal differentiation, and implicates RAB1A in the etiology of the neurodevelopmental sequelae associated with the 2p14-p15 microdeletion syndrome.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Genome-wide identification and expression analysis reveal the role of histone methyltransferase and demethylase genes in heat stress response in potato (Solanum tuberosum L.)

BackgroundPotato (Solanum tuberosum L.), the third most important non-cereal crop, is sensitive to high temperature. Histone modifications have been known to regulate various abiotic stress responses. However, the role of histone methyltransferases and demethylases remain unexplored in potato under heat stress. MethodsPotato genome database was used for genome-wide analysis of StPRMT and StHDMA gene families, which were further characterized by analyzing gene structure, conserved motif, domain organization, sub-cellular localization, promoter region and phylogenetic relationships. Additionally, expression profiling under high-temperature stress in leaf and stolon tissue of heat contrasting potato genotypes was done to study their role in response to high temperature stress. ResultsThe genome-wide analysis led to identification of nine StPRMT and eleven StHDMA genes. Structural analysis, including conserved motifs, exon/intron structure and phylogenetic relationships classified StPRMT and StHDMA gene families into two classes viz. Class I and Class II. A variety of cis-regulatory elements were explored in the promoter region associated with light, developmental, hormonal and stress responses. Prediction of sub-cellular localization of StPRMT proteins revealed their occurrence in nucleus and cytoplasm, whereas StHDMA proteins were observed in different sub-cellular compartments. Furthermore, expression profiling of StPRMT and StHDMA gene family members revealed genes responding to heat stress. Heat-inducible expression of StPRMT1, StPRMT3, StPRMT4 and StPRMT5 in leaf and stolon tissues of HS and HT cultivar indicated them as probable candidates for enhancing thermotolerance in potato. However, StHDMAs responded dynamically in leaf and stolon tissue of heat contrasting genotypes under high temperature. ConclusionThe current study presents a detailed analysis of histone modifiers in potato and indicates their role as an important epigenetic regulators modulating heat tolerance. General significanceUnderstanding epigenetic mechanisms underlying heat tolerance in potato will contribute towards breeding of thermotolerant potato varieties.

ListiWiki: A database for the foodborne pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive foodborne pathogen that regularly causes outbreaks of systemic infectious diseases. The bacterium maintains a facultative intracellular lifestyle; it thrives under a variety of environmental conditions and is able to infect human host cells. L. monocytogenes is genetically tractable and therefore has become an attractive model system to study the mechanisms employed by facultative intracellular bacteria to invade eukaryotic cells and to replicate in their cytoplasm. Besides its importance for basic research, L. monocytogenes also serves as a paradigmatic pathogen in genomic epidemiology, where the relative stability of its genome facilitates successful outbreak detection and elucidation of transmission chains in genomic pathogen surveillance systems. In both terms, it is necessary to keep the annotation of the L. monocytogenes genome up to date. Therefore, we have created the database ListiWiki (http://listiwiki.uni-goettingen.de/) which stores comprehensive information on the widely used L. monocytogenes reference strain EDG-e. ListiWiki is designed to collect information on genes, proteins and RNAs and their relevant functional characteristics, but also further information such as mutant phenotypes, available biological material, and publications. In its present form, ListiWiki combines the most recent annotation of the EDG-e genome with published data on gene essentiality, gene expression and subcellular protein localization. ListiWiki also predicts protein-protein interactions networks based on protein homology to Bacillus subtilis proteins, for which detailed interaction maps have been compiled in the sibling database SubtiWiki. Furthermore, crystallographic information of proteins is made accessible through integration of Protein Structure Database codes and AlphaFold structure predictions. ListiWiki is an easy-to-use web interface that has been developed with a focus on an intuitive access to all information. Use of ListiWiki is free of charge and its content can be edited by all members of the scientific community after registration. In our labs, ListiWiki has already become an important and easy to use tool to quickly access genome annotation details that we can keep updated with advancing knowledge. It also might be useful to promote the comprehensive understanding of the physiology and virulence of an important human pathogen.

Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumourigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumourigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin β-3, binding partner fibronectin-1, matrix metalloprotease enzymes promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a Chicken Embryo xenograft model for in vivo validation, we show that +CA MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional pro-tumorigenic alterations) is sufficient to confer early pro-tumourigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging.

CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

GIFTdb: a useful gene database for plant fruit traits improving.

Fruit traits are critical determinants of plant fitness, resource diversity, productive and quality. Gene regulatory networks in plants play an essential role in determining fruit traits, such as fruit size, yield, firmness, aroma and other important features. Many research studies have focused on elucidating the associated signaling pathways and gene interaction mechanism to better utilize gene resources for regulating fruit traits. However, the availability of specific database of genes related to fruit traits for use by the plant research community remains limited. To address this limitation, we developed the Gene Improvements for Fruit Trait Database (GIFTdb, http://giftdb.agroda.cn). GIFTdb contains 35 365 genes, including 896 derived from the FR database 1.0, 305 derived from 30 882 articles from 2014 to 2021, 236 derived from the Universal Protein Resource (UniProt) database, and 33 928 identified through homology analysis. The database supports several aided analysis tools, including signal transduction pathways, gene ontology terms, protein-protein interactions, DNAWorks, Basic Local Alignment Search Tool (BLAST), and Protein Subcellular Localization Prediction (WoLF PSORT). To provide information about genes currently unsupported in GIFTdb, potential fruit trait-related genes can be searched based on homology with the supported genes. GIFTdb can provide valuable assistance in determining the function of fruit trait-related genes, such as MYB306-like, by conducting a straightforward search. We believe that GIFTdb will be a valuable resource for researchers working on gene function annotation and molecular breeding to improve fruit traits.

Tracking Cell Wall-Anchored Proteins in Gram-Positive Bacteria.

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

Detection of Cell Wall-Anchoring Machinery by Immunogold-Labeling Thin-Section Electron Microscopy.

Cell wall anchoring of surface proteins and pili in Gram-positive bacteria is mediated by sortase - a highly conserved transpeptidase enzyme. Early studies have demonstrated the membrane-associated nature of this enzyme in close proximity with its cognate substrates, using immunogold-labeling thin-section electron microscopy. Here, we provide a detail protocol of this methodology, including specimen preparation, ultrathin sectioning, and immunogold-labeling electron microscopic procedures, with an experimental model of sortase enzymes from Actinomyces oris. In principle, this protocol can be employed for any bacterial ultrathin-section samples to detect subcellular localization of proteins and organelles by immuno-electron microscopy.

Probing Bacterial Cell Division and Cell Envelope Biogenesis with Live-Cell Fluorescence Microscopy.

The development of advanced microscopy techniques has ushered in a new era of research as it helps understand biological processes on a deeper, mechanistic, and molecular level like never before. Live-cell fluorescence microscopy has importantly allowed us to visualize subcellular protein localization and incorporation of various fluorophores compatible with living cells in real time. As such, this technique offers valuable insights at the single-cell level and enables us to monitor phenotypic differences that were easily overlooked at a population level. One area of research that has benefited greatly from these advances is the study of the bacterial cell envelope biogenesis and cell division process. In this report, we provide detailed protocols, optimized in our lab, for imaging these processes in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus.

The Identification of Common Druggable Targets For Acute Dysentery in Four Pathogenic Bacteria, Shigella, Salmonella, Campylobacter and E.Coli

Purpose: Dysentery is a severe form of diarrhea and caused by four bacteria include Shigella, Campylobacter, E.coli and Salmonella. They are responsible for higher morbidity and mortality rates resulting from dysentery every year across the world. Antibiotic therapy of this disease plays a critical role in decreasing the prevalence as well as the fatality rate of this infection. However, the management of this disease remains challenging, owing to the overall increase in resistance against many antimicrobials. Hence, it has become important to identify as well as develop therapeutic methods presenting novel avenues for infections. In the current study, proteome based mapping was utilized to find the potential drug targets for dysentery. There is need to identify novel drug and vaccine targets to control this disease. This study is designed to identify new drug targets to develop drug and vaccines to battle bacillary dysentery. Subtractive genomics approach was used in this study to find novel drug targets.&#x0D; Methodology: The proteomes of Shigella, E. coli, Campylobacter and Salmonella were retrieved from Uniprot. Paralogs in these proteomes were removed by CD-HIT. Gene essentiality was screened by Geptop server 2.0. Host-pathogen interaction was analyzed through HPIDB database. To identify non-homologous proteins, the essential proteins were analyzed in Blastp against Homo sapiens (H. sapiens). The unique metabolic pathways were recognized by comparison of metabolic pathways of selected strains of bacteria and H. sapiens using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Subcellular localization and functional classification of proteins involved in unique metabolic pathways were analyzed through PSORTb and SVMProt. Druggability potential of unique essential proteins was investigated using the DrugBank database.&#x0D; Findings: Over all analysis showed 7 proteins that were common in all four bacteria. These proteins were essential to pathogens. Out of 7 proteins 6 proteins MURA, MURG, DAPA, MURB, DAPE, DnaA are reported as a drug target in different bacteria and one protein DAPD is novel. The study will enable the development of natural and cost-effective drugs against dysentery infections.&#x0D; Recommendation: However, further validations are necessary to confirm their drug effectiveness and biocompatibility.

ERH gene knockdown inhibits the proliferation and migration of ARPE-19 cells through MCM complex and EMT process

PurposeTo explore the role of the Enhancer of rudimentary homolog (ERH) gene on the proliferation and migration of ARPE-19 cells, and its mechanism. MethodsARPE-19 cells were divided into ERH gene knockdown (ERH KD) and normal ERH gene (ERH NC) groups and infected with respected virus. Cell counting kit-8 assay, wound-healing assay, and flow cytometry were performed to evaluate the effects of the ERH gene on cell proliferation, migration, and cell cycle. A 4D label-free quantitative proteomic analysis was conducted to obtain the ERH gene knockdown-related differential proteins list (DPL). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein domain analysis, subcellular localization analysis, and protein–protein interaction (PPI) analysis were performed to explore the main downstream functions of the ERH gene. Proteins related to DNA replication, cell cycle, and epithelial-mesenchymal transition (EMT) were identified by Western blot test. ResultsThe ERH gene was successfully knocked down in ARPE-19 cells of the ERH KD group. The proliferation and migration of cells were reduced and the cell cycle was arrested at the S phase in the ERH KD group. A DPL of 47 upregulated and 108 downregulated proteins was obtained, and their functions were explored and found to be associated with the MCM complex, DNA replication, and cell cycle. Protein domain analysis, protein subcellular localization analysis, and PPI analysis showed that the MCM complex may play a key role in the proliferation of ARPE-19 cells affected by the ERH gene. DNA replication, cell cycle, and EMT-related proteins were affected when the ERH gene was knocked down. ConclusionKnockdown of ERH gene inhibits the proliferation and migration of ARPE-19 cells through the MCM complex and EMT process.

Novel variants in the CLCN4 gene associated with syndromic X-linked intellectual disability.

The dysfunction of the CLCN4 gene can lead to X-linked intellectual disability and Raynaud-Claes syndrome (MRXSRC), characterized by severe cognitive impairment and mental disorders. This study aimed to investigate the genetic defects and clinical features of Chinese children with CLCN4 variants and explore the effect of mutant ClC-4 on the protein expression level and subcellular localization through in vitro experiments. A total of 401 children with intellectual disabilities were screened for genetic variability using whole-exome sequencing (WES). Clinical data, including age, sex, perinatal conditions, and environmental exposure, were collected. Cognitive, verbal, motor, and social behavioral abilities were evaluated. Candidate variants were verified using Sanger sequencing, and their pathogenicity and conservation were analyzed using in silico prediction tools. Protein expression and localization of mutant ClC-4 were measured using Western blotting (WB) and immunofluorescence microscopy. The impact of a splice site variant was assessed with a minigene assay. Exome analysis identified five rare CLCN4 variants in six unrelated patients with intellectual disabilities, including two recurrent heterozygous de novo missense variants (p.D89N and p.A555V) in three female patients, and two hemizygous missense variants (p.N141S and p.R694Q) and a splicing variant (c.1390-12T > G) that are maternally inherited in three male patients. The p.N141S variant and the splicing variant c.1390-12(T > G were novel, while p.R694Q was identified in two asymptomatic heterozygous female patients. The six children with CLCN4 variants exhibited a neurodevelopmental spectrum disease characterized by intellectual disability (ID), delayed speech, autism spectrum disorders (ASD), microcephaly, hypertonia, and abnormal imaging findings. The minigene splicing result indicated that the c.1390-12T > G did not affect the splicing of CLCN4 mRNA. In vitro experiments showed that the mutant protein level and localization of mutant protein are similar to the wild type. The study identified six probands with CLCN4 gene variants associated with X-linked ID. It expanded the gene and phenotype spectrum of CLCN4 variants. The bioinformatic analysis supported the pathogenicity of CLCN4 variants. However, these CLCN4 gene variants did not affect the ClC-4 expression levels and protein location, consistent with previous studies. Further investigations are necessary to investigate the pathogenetic mechanism.