Protein Subcellular Localization Research Articles

Application of spatial metrology models in cell molecular localization and functional prediction

Understanding a protein’s exact cellular location is often essential to understanding its function. Even with the advancements in computer approaches, protein localization prediction indeed faces major obstacles such as interpretability and handling numerous localization sites. In this research, a novel approach, Squirrel Search Optimized Dynamic Visual Geometry Group Network (SSO-DVGG), is proposed to improve protein sub-cellular localization predictions by utilizing spatial metrology models to tackle these problems. With its simplified architecture, SSO-DVGG can explain whether a protein is directed to particular cellular sites, as well as identify important sequence components like sorting motifs or localization signals. This model allows users to select acceptable error levels by providing a confidence estimate for each prediction and highlighting sequence properties that are responsible for localization. This makes the model interpretable. Furthermore, SSO-DVGG uses a probabilistic methodology and integrates a large amount of data from dual-targeted proteins, which enables it to predict multiple localization locations per protein accurately. SSO-DVGG outperforms the best predictors and shows superior capacity to predict multiple localizations when tested on several independent datasets. By providing a clear and accurate understanding of protein distribution and function, this method promotes the application of spatial metrology models in cell molecular localization and functional prediction.

Small-Molecule Benzo-Phenoselenazine Derivatives for Multi-Subcellular Biomolecule Profiling.

Elucidating the subcellular localization of RNAs and proteins is fundamental to understanding their biological functions. Genetically encoded proteins/enzymes provide an attractive approach to target many proteins of interest, but are limited to specific cell lines. Although small-molecule-based methods have been explored, a comprehensive system for profiling multiple locations in living cells, comparable to fusion-protein techniques, is yet to be established. In this study, we introduce a novel proximity labeling strategy employing a suite of small molecules derived from benzophenoselenazine (e.g., selenium-containing Nile Blue [SeNB]), which achieves proximity labeling through singlet oxygen generation upon near-infrared light activation in the presence of propargylamine. These SeNB compounds allow forselective labeling of RNAs and proteins within living cells, exhibiting a distinct preference for organelle membranes, which are systematically investigated via in vitro, computational, and in cellulo examinations. Our findings highlight the capabilities of SeNB derivatives as wash-free and genetics-free approaches to illuminate the subcellular localization of biological molecules with deep penetration and high spatial resolution. Moreover, SeNB derivatives are capable of elucidating inter-organelle interactions at the molecular level, as evidenced by proteomic and transcriptomic analyses, thus holding significant potential for advancing our understanding of cellular processes related to disease progression and therapeutic development.

Phototactic signaling network in rod-shaped cyanobacteria: A study on Synechococcus elongatus UTEX 3055

Light-controlled motility is advantageous for photosynthetic prokaryotes to better survive in environment with constantly changing light conditions. For cyanobacteria, light is both an energy source for photosynthesis and a stress factor. Consequently, some cyanobacteria evolved the ability to control type-IV pili (T4P)-mediated surface motility using a chemotaxis-like system in response to light signals. Extensive studies on the mechanism of phototaxis has been conducted in the spherical Synechocystis sp. PCC 6803 and the filamentous strain Nostoc punctiforme, while less is explored in rod-shaped cyanobacteria such as Synechococcus species. In this study, we investigated the phototaxis pathway in the unicellular rod-shaped cyanobacterium Synechococcus elongatus UTEX 3055, which exhibits bidirectional phototaxis using a single tax1 operon, in contrast to more complex and multiple gene clusters revealed in Synechocystis sp. PCC 6803. Results obtained by protein-protein interaction assays and protein subcellular localization experiments indicated that proteins encoded by the tax1 operon form large clusters that asymmetrically distributed both between the two poles and within the same pole. In vitro phosphorylation assays and site-directed mutations of conserved phosphorylation sites in PixLSe, PixGSe and PixHSe demonstrate that PixLSe acts as a histidine kinase, and PixGSe and PixHSe as response regulators for signal transduction. We further show that PixGSe and PixHSe are recruited to cell poles via interactions with the N-terminal region of PixLSe. While phosphotransfer reactions in this signaling pathway are critical for phototactic signaling, the two response regulators appear to play different roles in the control of phototaxis. This study provides a framework for further investigation into the complex phototactic signaling network in rod-shaped cyanobacteria with clearly defined cell poles in contrast to round shaped Synechocystis species with virtual cells poles through light-lensing effect.

Identification and analysis of drought-responsive F-box genes in upland rice and involvement of OsFBX148 in ABA response and ROS accumulation

BackgroundUpland rice varieties exhibit significant genetic diversity and broad environmental adaptability, making them ideal candidates for identifying consistently expressed stress-responsive genes. F-box proteins typically function as part of the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes to precisely regulate gene expression and protein level, playing essential roles in the modulation of abiotic stress responses. Therefore, utilizing upland rice varieties for screening stress-responsive F-box genes is a highly advantageous approach.ResultsThrough mRNA-seq analysis in the Brazilian upland rice (cv. IAPAR9), the research identified 29 drought-responsive F-box genes. Gene distribution and duplication analysis revealed these genes are distributed on 11 of the 12 chromosomes and 10 collinear gene pairs were identified on different chromosomes. 13 cis-elements or binding sites were identified in the promoters of the 29 drought-responsive F-box genes by analysis. Protein domain, stability and subcellular localization analysis results suggest that these F-box proteins possess F-box domain and several other domains, and they are mostly unstable proteins with subcellular localization in cytoplasm, nucleus, chloroplasts, mitochondria and endoplasmic reticulum. Most of drought-responsive F-box genes exhibited expression in various tissues such as root, stem, leaf, leaf sheath and panicle except for OsFBO10 and OsFBX283. These genes exhibited various responses to abiotic stresses such as osmotic, cold, heat, and salt stresses, along with ABA treatment. Importantly, a frame-shift mutation in OsFBX148 was created in the ZH11 variety, leading to altered ABA signal transduction and ROS accumulation. The study further elucidated the interaction of OsFBX148 with SKP1 family proteins OSK4/7/17 to form the SCF complex, dependent on the F-box domain.ConclusionsThe research identified and analyzed 29 drought-responsive F-box genes in upland rice and provides valuable insights into the role of OsFBX148 in ABA and ROS responses. It establishes a basis for future exploration of F-box genes in improving resistance to abiotic stresses, especially drought.

Impact of Alignments on the Accuracy of Protein Subcellular Localization Predictions.

Alignments in bioinformatics refer to the arrangement of sequences to identify regions of similarity that can indicate functional, structural, or evolutionary relationships. They are crucial for bioinformaticians as they enable accurate predictions and analyses in various applications, including protein subcellular localization. The predictive model used in this article is based on a deep - convolutional architecture. We tested configurations of Deep N-to-1 convolutional neural networks of various depths and widths during experimentation for the evaluation of better-performing values across a diverse set of eight classes. For without alignment assessment, sequences are encoded using one-hot encoding, converting each character into a numerical representation, which is straightforward for non-numerical data and useful for machine learning models. For with alignments assessment, multiple sequence alignments (MSAs) are created using PSI-BLAST, capturing evolutionary information by calculating frequencies of residues and gaps. The average difference in peak performance between models with alignments and without alignments is approximately 15.82%. The average difference in the highest accuracy achieved with alignments compared with without alignments is approximately 15.16%. Thus, extensive experimentation indicates that higher alignment accuracy implies a more reliable model and improved prediction accuracy, which can be trusted to deliver consistent performance across different layers and classes of subcellular localization predictions. This research provides valuable insights into prediction accuracies with and without alignments, offering bioinformaticians an effective tool for better understanding while potentially reducing the need for extensive experimental validations. The source code and datasets are available at http://distilldeep.ucd.ie/SCL8/.

Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus.

Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus.

A genome-engineered tool set for Drosophila TGF-β/BMP signaling studies.

Ligands of the TGF-β/BMP superfamily are crucially involved in the regulation of growth, patterning and organogenesis and can act as long-range morphogens. Essential for understanding TGF-β/BMP signaling dynamics and regulation are tools that allow monitoring and manipulating pathway components at physiological expression levels and endogenous spatiotemporal patterns. We used genome engineering to generate a comprehensive library of endogenously epitope- or fluorescent-tagged versions of receptors, co-receptors, transcription factors and key feedback regulators of the Drosophila BMP and Activin signaling pathways. We demonstrate that the generated alleles are biologically active and can be used for assessing tissue and subcellular distribution of the corresponding proteins. Furthermore, we show that the genomic platforms can be used for in locus structure-function and cis-regulatory analyses. Finally, we present a complementary set of protein binder-based tools, which allow visualization as well as manipulation of the stability and subcellular localization of epitope-tagged proteins, providing new tools for the analysis of BMP signaling and beyond.

Predicting the subcellular location of prokaryotic proteins with DeepLocPro.

Protein subcellular location prediction is a widely explored task in bioinformatics because of its importance in proteomics research. We propose DeepLocPro, an extension to the popular method DeepLoc, tailored specifically to archaeal and bacterial organisms. DeepLocPro is a multiclass subcellular location prediction tool for prokaryotic proteins, trained on experimentally verified data curated from UniProt and PSORTdb. DeepLocPro compares favorably to the PSORTb 3.0 ensemble method, surpassing its performance across multiple metrics in our benchmark experiment. The DeepLocPro prediction tool is available online at https://ku.biolib.com/deeplocpro and https://services.healthtech.dtu.dk/services/DeepLocPro-1.0/. Supplementary data are available at Bioinformatics online.

DNAR-12. STEAROYL-COA DESATURASE REGULATES DNA DAMAGE REPAIR IN GBM BY MODULATING PARP1 ACTIVITY

Abstract Glioblastoma (GBM) is the most prevalent malignant brain cancer in adults, notorious for its aggressiveness and resistance to therapy. GBM patients commonly receive radiation therapy combined with temozolomide (TMZ), which engages tumor cells to promote lethal DNA damage and subsequent cytotoxicity. Active DNA repair mechanisms, particularly in a subset population of GBM stem-like cells (GSCs), drive therapeutic resistance, thus largely contributing to the bleak prognosis of GBM. Poly (ADP-ribose) polymerases (PARPs) are essential in various cellular processes, including DNA repair. We have previously reported that fatty acid desaturation mediated by Stearoyl-CoA desaturase 1 (SCD1) plays a cytoprotective role in GSCs. Inhibiting SCD prevents tumor initiation and inhibits DNA damage repair, hence sensitizing to radiation and TMZ. Yet, the mechanistic role of SCD in DNA damage regulation remained unknown. Here, we found that the expression of both human isoforms of SCD, SCD1, and SCD5, directly correlates with PARP protein levels and subcellular localization. Downregulating SCD1/5 in GSCs resulted in significantly lower levels of PARP1, coupled with increased DNA damage, as well as reduced DNA damage repair and chromosome assembly. Conversely, cells overexpressing SCD1/5 exhibited higher PARP1 expression and lower levels of basal DNA damage compared to control GSCs. Notably, SCD1/5 overexpression also led to increased resistance to TMZ. We show that SCD1/5 regulates PARP activity by controlling PARP1 subcellular localization. In addition to decreased total levels of PARP following SCD1/5 downregulation, we also found increased PARP1 self-PARylation. Using confocal microscopy, we show that when SCD1/5 is repressed, PARylated PARP translocates from the nucleus to the cytosol, rendering it unable to bind DNA and recruit DNA-repairing machinery. In conclusion, our study provides a direct link between fatty acid metabolism and PARP1-mediated DNA damage repair.

Kidney mRNA-protein expression correlation: what can we learn from the Human Protein Atlas?

The Human Protein Atlas, with more than 10 million immunohistochemical images showing tissue- and cell-specific protein expression levels and subcellular localization information, is widely used in kidney research. The Human Protein Atlas contains comprehensive data on multi-tissue transcript and protein abundance, allowing for comparisons across tissues. However, while visual and intuitive to interpret, immunohistochemistry is limited by its semi-quantitative nature. This can lead to mismatches in protein expression measurements across different platforms. We performed a comparison of the Human Protein Atlas' kidney-specific RNA sequencing and immunohistochemistry data to determine whether the mRNA and protein abundance levels are concordant. Our study shows that there is a discordance between mRNA and protein expression in the kidney based on the Human Protein Atlas data. Using an external validation mass spectrometry dataset, we show that more than 500 proteins undetected by immunohistochemistry are robustly measured by mass spectrometry. The Human Protein Atlas transcriptome data, on the other hand, exhibit similar transcript detection levels as other kidney RNA-seq datasets. Discordance in mRNA-protein expression could be due to both biological and technical reasons, such as transcriptional dynamics, translation rates, protein half-lives, and measurement errors. This is further complicated by the heterogeneity of the kidney tissue itself, which can increase the discordance if the cell populations or tissue compartment samples do not match. As such, shedding light on the mRNA-protein relationship of the kidney-specific Human Protein Atlas data can provide context to our scientific inferences onrenal gene and protein quantification.

Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy

We have developed a method combining microinjection and automated fluorescence microscopy to continuously assess the degradation rate, subcellular localization and intracellular concentration of protein analytes at the single-cell level. Cells are unperturbed and grown in unaltered environmental conditions and show high viability. The injection of analytes at defined ratios and concentrations allows for a clearly defined starting point of degradation, without the entanglement of biosynthesis/uptake, often encountered in existing methods. The possibility to evaluate, add, or remove post-translational modifications prior to injection represents a powerful tool to assess minute protein degradation rate changes with high precision and allowed us to determine the absolute degradation rates caused by N-degron pathway engagement, with a focus on the role of acetylation. The low degradation rate of eGFP was found to be caused by inefficient N-terminal proteasomal unfolding. We moreover quantified the surprisingly strong influences of commonly used peptide tags and detected high variation between fluorescent proteins with regard to both protein degradation and subcellular localization. Furthermore, we have validated the use of chemically coupled dyes as robust reporters for protein degradation, and elucidated the significance of their membrane-permeability, thereby extending the applicability of our method to any protein of interest.

Rab11 suppresses neuronal stress signaling by localizing dual leucine zipper kinase to axon terminals for protein turnover.

Dual leucine zipper kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals for protein turnover and a failure of such transport can lead to neuronal loss. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling.

Proteogenomic Approaches for Diseasome Studies.

During the last three decades, technological advancements in high-throughput next-generation sequencing have resulted in an increased understanding of proteomic and genomic data, aptly termed proteogenomics. Efforts in developing such approaches have not only been limited but also focused on protein identification and subcellular localization. These approaches, however, have also been explored for their broad understanding of how genomics/transcriptomics data have yielded measures, for example, gene expression regulation/signal cascading and diseasome studies. In this review, we discuss methods and tools developed through sequence-centric integrative modeling of proteogenomic approaches.

Autographa californica multiple nucleopolyhedrovirus ac106 is required for the nuclear egress of nucleocapsids and intranuclear microvesicle formation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf106 (ac106) is highly conserved in baculoviruses. Previous studies have shown that ac106 is required for the production of infectious budded virions (BVs). However, the functional role of ac106 in virion morphogenesis remains unknown. In this report, an ac106 knockout virus and an ac106 repair virus were constructed. The effect of ac106 deletion on virion morphogenesis was investigated, and the expression and subcellular localization of the Ac106 protein were characterized. Our data indicated that ac106 is required for the nuclear egress of nucleocapsids and intranuclear microvesicle formation, as well as subsequent BV and occlusion-derived virion (ODV) production and the embedding of ODVs into polyhedra. Ac106 is a baculovirus late protein that is concentrated in discrete foci of virus-induced membrane structures in the intranuclear ring zone of virus-infected cells. Further studies on the relationship between Ac106 and four other proteins that are also required for intranuclear microvesicle formation, Ac75, Ac76, Ac93, and P48 (Ac103), revealed that Ac106 is associated with Ac75, Ac76, Ac93, P48, and itself. Ac106 is required for Ac75, Ac93, and P48 accumulation in foci of virus-induced intranuclear membrane structures and the intranuclear transport of Ac76. Analysis of the subcellular localization of ODV integral envelope proteins upon deletion of the genes required for intranuclear microvesicle formation indicated that intranuclear microvesicle formation may be essential for ODV integral envelope protein transport into the nucleus, supporting the hypothesis that intranuclear microvesicles originate from the nuclear membrane.IMPORTANCEBaculovirus occlusion-derived virions (ODVs) are known to acquire their envelopes from virus-induced intranuclear microvesicles within the nucleoplasm, and this strategy of intranuclear envelopment of nucleocapsids to form virions is unique among viruses. However, the mechanism of ODV morphogenesis, particularly intranuclear microvesicle formation, remains unclear. In this study, we identified ac106 as the fifth gene, in addition to ac75, ac76, ac93, and p48 (ac103), which are required for intranuclear microvesicle formation. Further studies on the relationship between ac106 and the other four genes, as well as the effect of ac106 or ac75 deletion on the localization of ODV integral envelope proteins, indicated that intranuclear microvesicle formation may be essential for the transport of ODV integral envelope proteins into the nucleus, which strongly supports the hypothesis that intranuclear microvesicles originate from the nuclear membrane. These findings greatly enhance our understanding of the molecular mechanism of baculovirus ODV morphogenesis.

The important role of the Wnt/β-catenin signaling pathway in small molecules mediated gingival mesenchymal stem cells transdifferentiate into neuron-like cells

ObjectiveGiven their neural crest origin, gingival mesenchymal stem cells (GMSCs) possess high neurogenic potential, which makes them suitable for cell replacement therapy against neurodegenerative diseases. This study investigated whether GMSCs can be transdifferentiated into neurons in vitro using a protocol involving small molecules VCRFY (VPA, CHIR99021, Repsox, Forskolin, and Y-27632). The regulatory mechanisms of key signaling pathways were also investigated. MethodsNeuronal induction of GMSCs was conducted using a small molecules-based protocol over 7 days, which included the evaluation of cell morphology, proliferation, expressions of neurogenic markers, and intracellular calcium oscillation. The activation of canonical the Wnt signaling pathway was assessed by examining the protein content and subcellular localization of β-catenin. ResultsSmall molecules-treated GMSCs displayed neuronal morphology and increased expression of neurogenic markers, including class III beta-tubulin (TUJ1), neuron-specific enolase (NSE), microtube-associated protein 2 (MAP2), and neurofilament medium (NFM), verified through RT-qPCR, western blotting, and immunocytochemistry. Based on the results of Fluo-4 AM calcium flux assay, small molecules-treated GMSCs exhibited enhanced electrophysiological activity. GMSC proliferation halted after 2 days of treatment. Among the small molecules, CHIR99021 exhibited the highest neuronal induction efficiency. Furthermore, activation of the Wnt/β-catenin signaling pathway augmented neuronal differentiation. ConclusionsSmall molecule-based cellular reprogramming can efficiently generate neurons from GMSCs, with Wnt/β-catenin signaling to play a critical role in neuronal induction.

Generation of four human-derived iPSC TorsinA-3xFLAG reporter lines from a DYT-TOR1A patient

A 3-bp deletion (ΔGAG) in TOR1A is a common cause of early-onset isolated dystonia DYT-TOR1A. The exact disease mechanism remains unknown. Here we describe the generation and characterization of four TorsinA-3xFLAG reporter induced pluripotent cell (iPSC) lines derived from a DYT-TOR1A patient. The cell lines carry either a ΔGAG variant or a corrected allele and a mono- or biallelic 3xFLAG-Tag introduced using CRISPR/Cas9 technology. These cells provide an opportunity to study differential protein stability, subcellular localization, and interactors of endogenous WT or ΔE variants of TorsinA in iPSCs, neural progenitor cells (smNPC), and neurons.

Cloning and Expression Analysis of ATG8 (Autophagy-Related 8) Gene Family in Solanaceae.

The autophagy-related gene family ATG8 (Autophagy-related 8) plays an important role in plant growth, development, and stress response. In this study, 15 ATG8 gene family sequences were amplified from Solanaceae, namely tobacco, tomato, and pepper, using RT-PCR to evaluate their basic properties, protein structure, and function, as well as the role of ATG8 in autophagy. The physicochemical properties, the predicted secondary and tertiary protein structures, subcellular localisation, gene structures, conserved motifs, and phylogenetic relationships of the ATG8 genes were analysed using bioinformatic techniques, and their expression patterns under sericin-induced plant disease resistance were investigated by RT-qPCR. The lengths of these proteins ranged from 79 to 120 aa, while their predicted molecular weights and isoelectric points (PI) ranged from 9283.62 to 13,778.74 and 6.32 to 11.44, respectively. The majority of the proteins were localised in the nucleus or chloroplasts. Conserved protein motifs and various cis-regulatory elements in the protein, with a wide range of related functions, were identified. The ATG8 gene family members showed expression changes after treatment with osthole, which induces disease resistance in tobacco, tomato, and pepper. These findings provide a foundation for further analyses of the ATG8 gene family in Solanaceae and the mechanism underlying the response to adverse conditions.

Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.

Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell-cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.

Biochemical Properties of Synaptic Proteins Are Dependent on Tissue Preparation: NMDA Receptor Solubility Is Regulated by the C-Terminal Tail.

Synaptic proteins are essential for neuronal development, synaptic transmission, and synaptic plasticity. The postsynaptic density (PSD) is a membrane-associated structure at excitatory synapses, which is composed of a huge protein complex. To understand the interactions and functions of PSD proteins, researchers have employed a variety of imaging and biochemical approaches including sophisticated mass spectrometry. However, the field is lacking a systematic comparison of different experimental conditions and how they might influence the study of the PSD interactome isolated from various tissue preparations. To evaluate the efficiency of several common solubilization conditions, we isolated receptors, scaffolding proteins, and adhesion molecules from brain tissue or primary cultured neurons or human forebrain neurons differentiated from induced pluripotent stem cells (iPSCs). We observed some striking differences in solubility. We found that N-methyl-d-aspartate receptors (NMDARs) and PSD-95 are relatively insoluble in brain tissue, cultured neurons, and human forebrain neurons compared to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs) or SAP102. In general, synaptic proteins were more soluble in primary neuronal cultures and human forebrain neurons compared to brain tissue. Interestingly, NMDARs are relatively insoluble in HEK293T cells suggesting that insolubility does not directly represent the synaptic fraction but rather it is related to a detergent-insoluble fraction such as lipid rafts. Surprisingly, truncation of the intracellular carboxyl-terminal tail (C-tail) of NMDAR subunits increased NMDAR solubility in HEK293T cells. Our findings show that detergent, pH, and temperature are important for protein preparations to study PSD protein complexes, and NMDAR solubility is regulated by its C-tail, thus providing a technical guide to study synaptic interactomes and subcellular localization of synaptic proteins.

5105 Characterization Of Novel PHEX Variants In X-Linked Hypophosphatemic Rickets And Genotype-PHEX Activity Correlation

Abstract Disclosure: H. Wu: None. W. Zhao: None. X. Chen: None. L. Gao: None. C. Xu: None. J. Zhao: None. X-linked hypophosphatemia (XLHR) is the most common genetic form of hypophosphatemic rickets (HR), which is caused by mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Till now, the genotype-phenotype relationship of XLHR and the pathogenic role of PHEX have not been fully determined. Here we summarized clinical features of 49 HR patients and detected 17 novel PHEX variants and 5 novel non-PHEX variants. Then we explored the pathogenesis of new-found variants from protein expression, glycosylation analysis, subcellular localization and endopeptidase activity. The results showed that missense variants (Q189H and X750R) slightly reduced protein expression without obviously altering the length and localization of the protein, while truncating variants significantly impaired the synthesis of PHEX and produced a shorter immature protein retained in cells. No evident correlation was observed between mutation types and clinical phenotypes. However, for the first time ever, we analyzed the relationship between PHEX activity and serum phosphorus level and found that patients with low PHEX activity tended to have severe hypophosphatemia and high rickets severity score (RSS). Then we established two new knock-in XLHR mouse models by introducing into mice two novel Phex variants (c.T1349C and c.C426G) found in patients using CRISPR/Cas9 technology. Both demonstrated the clinical manifestations of XLHR seen in the patients and PhexC426G mice showed more severe phenotype than PhexT[1]349C mice, which further confirmed the rationality of genotype-PHEX enzymatic activity correlation analysis. Therefore, our findings demonstrated that novel PHEX variants could disrupt protein function via affecting protein synthesis, post-translational modification, cellular trafficking and catalytic activity. Our study promoted the understanding of XLHR pathogenic mechanism and PHEX activity-phenotype correlation provided scientific support for precise diagnosis and treatment of XLHR. Presentation: 6/3/2024