Localization Of Proteins Research Articles

Importin α inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture. As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites). Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested. Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation. As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs.

Polar localization and local translation of RHO-RELATED PROTEIN FROM PLANTS2 mRNAs promote root hair growth in Arabidopsis.

Root hairs are tip-growing cells that anchor plants in the soil and are critical for water uptake, nutrient acquisition, and plant-environment interactions. While the molecular mechanisms that maintain the polar growth of root hairs through the asymmetric distribution of proteins, such as RHO-RELATED PROTEIN FROM PLANTS 2 (ROP2), have been described, it is unclear whether and how the transcripts encoding these tip-localized proteins are polarly localized and locally translated. Here, we demonstrated that ROP2 mRNA exhibits polar localization in Arabidopsis (Arabidopsis thaliana) root hairs. We showed that region VI (250-350 bp downstream of the stop codon) of the ROP2 3' untranslated region (UTR) is necessary for proper mRNA localization. Moreover, region VI-mediated ROP2 mRNA polar localization was required for local translation of ROP2 transcripts, contributing to the proper subcellular localization of ROP2. Region III (100-200 bp downstream of the stop codon) influenced the local translation of ROP2 mRNA. Phenotypic investigations demonstrated that both regions III and VI of the ROP2 3' UTR play crucial roles in modulating root hair growth. These findings help explain the local protein biosynthesis of ROP2, advancing our understanding of the regulatory mechanism and genetic basis of mRNA localization and local translation in plants.

Identification of novel germline mutations in FUT7 and EXT1 linked with hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder primarily linked with mutations in Exostosin-1 (EXT1) and Exostosin-2 (EXT2) genes. However, not all HME cases can be explained by these mutations, and its pathogenic mechanisms are not fully understood. Herein, utilizing whole-exome sequencing and genetic screening with a family trio design, we identify two novel rare mutations co-segregating with HME in a Chinese family, including a nonsense mutation (c.204G>A, p.Trp68*) in EXT1 and a missense mutation (c.893T>G, p.Phe298Cys) in FUT7. Functional assays reveal that the FUT7 mutation affects the cellular localization of FUT7 protein and regulates cell proliferation. Notably, the simultaneous loss of fut7 and ext1 in a zebrafish model results in severe chondrodysplasia, indicating a functional link between FUT7 and EXT1 in chondrocyte regulation. Additionally, we unveil that FUT7 p.Phe298Cys reduces EXT1 expression through IL6/STAT3/SLUG axis at the transcription level and through ubiquitination-related proteasomal degradation at the protein level. Together, our findings not only identify novel germline mutations in FUT7 and EXT1 genes, but also highlight the critical interaction between these genes, suggesting a potential 'second-hit' mechanism over EXT1 mutations in HME pathogenesis. This insight enhances our understanding of the mechanisms underlying HME and opens new avenues for potential therapeutic interventions.

An Alkyne-Containing Isoprenoid Analogue Based on a Farnesyl Diphosphate Scaffold Is a Biologically Functional Universal Probe for Proteomic Analysis.

Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

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.

Conserved and Unique Mitochondrial Target Sequence of TRPV4 Can Independently Regulate Mitochondrial Functions.

Though mitochondria have their own genome and protein synthesis machineries, the majority of the mitochondrial proteins are actually encoded by the nuclear genome. Most of these mitochondrial proteins are imported into specific compartments of the mitochondria due to their mitochondrial target sequence (MTS). Unlike the nuclear target sequence, the MTS of most of the mitochondrial localized proteins remain poorly understood, mainly due to their variability, heterogeneity, unconventional modes of action, mitochondrial potential-dependent transport, and other complexities. Recently, we reported that transient receptor potential vanilloid subtype 4 (TRPV4), a thermosensitive cation channel, is physically located at the mitochondria. Here we characterize a small segment (AA 592-630) located at the TM4-loop4-TM5 segment of TRPV4 that acts as a novel MTS. The same region remains highly conserved in all vertebrates and contains a large number of point mutations each of which causes an diverse spectrum of diseases in human. Using confocal and super-resolution microscopy, we show that this MTS of TRPV4 or its mutants localizes to the mitochondria independently and also induces functional and quantitative changes in the mitochondria. By using conformal microscopy, we could detect the presence of the MTS region within the isolated mitochondria. These findings may be important to understand the complexity of MTS and TRPV4-induced channelopathies better.

Mitochondrial targeting of Candida albicans SPFH proteins and requirement of stomatins for SDS-induced stress tolerance.

Stomatins and prohibitins coordinate respiration and stress adaptation in fungi. Invasive mycoses caused by Candida albicans are a significant cause of morbidity, and candidemia patients show high mortality rates worldwide. Mitochondria are essential for C. albicans commensalism and virulence, and mitochondrial proteins are targets for antifungal interventions. C. albicans encodes five SPFH proteins: two stomatin-like proteins and three prohibitins. We have previously shown that Slp3 is important for C. albicans adaptation to various types of environmental stress. Moreover, synthetic compounds that bind to mammalian prohibitins inhibit C. albicans filamentation and are fungicidal. However, there is limited information available regarding the remaining SPFH proteins. Our findings show that mitochondrial localization of SPFH proteins is conserved in C. albicans. In addition, we demonstrate the importance of stomatins in plasma membrane and mitochondrial stress tolerance.

Insights into the regulation of mRNA translation by scaffolding proteins.

Regionalisation of molecular mechanisms allows cells to fine-tune their responses to dynamic environments. In this context, scaffolds are well-known mediators of localised protein activity. These phenomenal proteins act as docking sites where pathway components are brought together to ensure efficient and reliable flow of information within the cell. Although scaffolds are mostly understood as hubs for signalling communication, some have also been studied as regulators of mRNA translation. Here, we provide a brief overview of the work unravelling how scaffolding proteins facilitate the cross-talk between the two processes. Firstly, we examine the activity of AKAP1 and AKAP12, two signalling proteins that not only have the capacity to anchor mRNAs to membranes but can also regulate protein synthesis. Next, we review the studies that uncovered how the ribosome-associated protein RACK1 orchestrates translation initiation. We also discuss the evidence pointing to the scaffolds Ezrin and LASP1 as regulators of early translation stages. In the end, we conclude with some open questions and propose future directions that will bring new insights into the regulation of mRNA translation by scaffolding proteins.

Modified Tou Nong Powder Obstructs Ulcerative Colitis by Regulating Autophagy and Mitochondrial Function.

Modified Tou Nong Powder (MTNP) is a traditional Chinese medicine formula widely used for treating body surface ulcers. Since colonic ulcers share similar pathological characteristics, MTNP has shown promising results in alleviating ulcerative colitis (UC) and has been safely used in clinical practice. This study aims to investigate how MTNP alleviates experimental colitis by inducing autophagy through the regulation of the AMP-activated protein kinase (AMPK)/Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) signaling pathway. In this study, UC rat models were created using 2,4,6-Trinitrobenzenesulfonic acid (TNBS). The therapeutic effects of MTNP on TNBS-induced colitis were evaluated through various methods such as disease activity index, visual examination, and histological examination of the colon. An inflammation model was also established in Caco-2 cells using H2O2. Western blot analysis was used to assess the expression of autophagy-related proteins, while immunofluorescence detection was employed for protein localization. Furthermore, quantitative real-time polymerase chain reaction (qPCR) was performed to analyze the expression of autophagy-related genes, confirming the role of MTNP in modulating the AMPK/PGC-1α signaling pathway. In vivo, oral administration of MTNP led to a remarkable reduction in colonic injury, inhibition of inflammatory infiltration, and improvement in the abnormal expression of inflammatory factors in colonic tissues. Furthermore, MTNP stimulated autophagy by activating the AMPK/PGC-1α signaling pathway, thereby mitigating mitochondrial dysfunction. In vitro, exposure to MTNP drug-containing serum (MTNP-DS) resulted in a reduction of reactive oxygen species levels, improvement in mitochondrial membrane potential, and activation of the AMPK/PGC-1α pathway, leading to the promotion of mitochondrial autophagy. The results indicate that MTNP triggers autophagy and enhances mitochondrial function, leading to the alleviation of UC in both in vitro and in vivo. These benefits are strongly linked to the activation of the AMPK/PGC-1α signaling pathway.

Understanding the molecular diversity of synapses.

Synapses are composed of thousands of proteins, providing the potential for extensive molecular diversity to shape synapse type-specific functional specializations. In this Review, we explore the landscape of synaptic diversity and describe the mechanisms that expand the molecular complexity of synapses, from the genotype to the regulation of gene expression to the production of specific proteoforms and the formation of localized protein complexes. We emphasize the importance of examining every molecular layer and adopting a systems perspective to understand how these interconnected mechanisms shape the diverse functional and structural properties of synapses. We explore current frameworks for classifying synapses and methodologies for investigating different synapse types at varying scales, from synapse-type-specific proteomics to advanced imaging techniques with single-synapse resolution. We highlight the potential of synapse-type-specific approaches for integrating molecular data with cellular functions, circuit organization and organismal phenotypes to enable a more holistic exploration of neuronal phenomena across different scales.

High-Throughput Single-Cell Analysis of Local Nascent Protein Deposition in 3D Microenvironments via Extracellular Protein Identification Cytometry (EPIC).

Extracellular matrix (ECM) guides cell behavior and tissue fate. Cell populations are notoriously heterogeneous leading to large variations in cell behavior at the single-cell level. Although insights into population heterogeneity are valuable for fundamental biology, regenerative medicine, and drug testing, current ECM analysis techniques only provide either averaged population-level data or single-cell data from a limited number of cells. Here, extracellular protein identification cytometry (EPIC) is presented as a novel platform technology that enables high-throughput measurements of local nascent protein deposition at single-cell level. Specifically, human primary chondrocytes are microfluidically encapsulated in enzymatically crosslinked microgels of 16 picoliter at kHz rates, forming large libraries of discrete 3D single-cell microniches in which ECM can be deposited. ECM proteins are labeled using fluorescence immunostaining to allow for nondestructive analysis via flow cytometry. This approach reveals population heterogeneity in matrix deposition at unprecedented throughput, allowing for the identification and fluorescent activated cell sorting-mediated isolation of cellular subpopulations. Additionally, it is demonstrated that inclusion of a second cell into microgels allows for studying the effect of cell-cell contact on matrix deposition. In summary, EPIC enables high-throughput single-cell analysis of nascent proteins in 3D microenvironments, which is anticipated to advance fundamental knowledge and tissue engineering applications.

Kinesin-like motor protein KIF23 maintains neural stemand progenitor cell pools in the developing cortex.

Accurate mitotic division of neural stem and progenitor cells (NSPCs) is crucial for the coordinated generation of progenitors and mature neurons, which determines cortical size and structure. While mutations in the kinesin-like motor protein KIF23 gene have been recently linked to microcephaly in humans, the underlying mechanisms remain elusive. Here, we explore the pivotal role of KIF23 in embryonic cortical development. We characterize the dynamic expression of KIF23 in the cortical NSPCs of mice, ferrets, and humans during embryonic neurogenesis. Knockdown of Kif23 in mice results in precocious neurogenesis and neuronal apoptosis, attributed to an accelerated cell cycle exit, likely resulting from disrupted mitotic spindle orientation and impaired cytokinesis. Additionally, KIF23 depletion perturbs the apical surface structure of NSPCs by affecting the localization of apical junction proteins. We further demonstrate that the phenotypes induced by Kif23 knockdown are rescued by introducing wild-type human KIF23, but not by a microcephaly-associated variant. Our findings unveil a previously unexplored role of KIF23 in neural stem and progenitor cell maintenance via regulating spindle orientation and apical structure in addition to cytokinesis, shedding light on microcephaly pathogenesis.

Interaction Between the Matrix Protein and the Polymerase Complex of Respiratory Syncytial Virus

The global burden of respiratory syncytial virus (RSV) and severe associated disease is prodigious. RSV-specific vaccines have been launched recently but there is no antiviral medicine commercially available. RSV polymerase (L) protein is one of the promising antiviral targets, along with fusion and nucleocapsid proteins. During medicinal chemistry campaigns, two potent L-protein inhibitors (PC786 and PC751) were identified. Both compounds inhibited the RSV A/B-induced cytopathic effect in HEp-2 cells equally, but PC786 was more potent than PC751 in bronchial epithelial cells. Repeated treatment with escalating concentrations on RSV A2-infected HEp-2 cells revealed both inhibitors led to a Y1631H mutation in the L protein, but only PC786 induced a mutation in the M protein (V153A). By L protein fragment and M protein binding analysis, we showed that the M protein interacts with the 1392–1735 amino acid region of the L protein, where PC786 potentially binds. In addition, PC786 treatment or PC786-induced mutant RSV was found to increase M-protein nuclear localisation later in infection, concomitant with delayed fusion protein localisation at the budding viral filaments. As M protein is known to play a key role in virus assembly and budding late in infection, our data suggests that disrupting the interaction between the M and L proteins could provide a novel target for antiviral development.

Spc2 modulates substrate- and cleavage site-selection in the yeast signal peptidase complex.

Secretory proteins are critically dependent on the correct processing of their signal sequence by the signal peptidase complex (SPC). This step, which is essential for the proper folding and localization of proteins in eukaryotic cells, is still not fully understood. In eukaryotes, the SPC comprises four evolutionarily conserved membrane subunits (Spc1-3 and Sec11). Here, we investigated the role of Spc2, examining SPC cleavage efficiency on various models and natural signal sequences in yeast cells depleted of or with mutations in Spc2. Our data show that discrimination between substrates and identification of the cleavage site by SPC is compromised when Spc2 is absent or mutated. Molecular dynamics simulation of the yeast SPC AlphaFold2-Multimer model indicates that membrane thinning at the center of SPC is reduced without Spc2, suggesting a molecular explanation for the altered substrate recognition properties of SPC lacking Spc2. These results provide new insights into the molecular mechanisms by which SPC governs protein biogenesis.

A nested case-control study of circular ribonucleic acid expression profiles in the peripheral blood of pregnant women with pre-eclampsia.

To examine the expression of circular ribonucleic acid (circRNA) in the peripheral blood of pregnant women with preeclampsia (PE) prior to the onset of the disease. A prospective nested case-control study included pregnant women who delivered at Fujian Maternal and Child Health Hospitals between November 2021 and October 2022. The study included three women diagnosed with PE (the PE group) and three healthy pregnant women (the control group). The expression of circRNAs in the peripheral blood before clinical diagnosis at 20 weeks gestation was analyzed by microarray technology, and the functional analysis and regulatory network prediction were done. There were 162 circRNAs with differentially expressed multiplicity of > 2.0 in the PE group compared to the control group. Out of them, 30 circRNAs were up-regulated and 132 were down-regulated. Gene ontology (GO) analysis revealed that the host genes of the differentially expressed circRNAs mainly participated in protein localization to plasma membrane, insemination, etc. Most enriched terms were related to cytoplasmic dynein complex, microtubule cytoskeleton, etc. In terms of molecular function, the enriched terms were related to dynein light intermediate chain binding, polyubiquitin modification-dependent protein binding, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis revealed that differentially expressed circRNAs were involved in vasopressin-regulated water reabsorption, cyclic guanosine monophosphate-protein kinase G (cGMP-PKG), calcium- and other signaling pathways. CircRNAs are involved in a variety of biological processes and regulatory network related to the pathogenesis of PE.

Multi-omics analysis reveals the neuroprotective effect of extract against Parkinson's disease in mouse.

To assess Atractylodis Rhizoma Alba extract (ARE) neuroprotective function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and related genes. Examined mRNA-DNA methylation changes induced by ARE in MPTP-induced Parkinson's disease (PD) model's substantia nigra. ARE mitigated MPTP-induced motor impairment in rotarod and open field tests and preserved tyrosine hydroxylase-positive neuronal cells in substantia nigra and striatum. Genome RNA-Sequencing and Methyl-Sequencing in substantia nigra of vehicle/ARE-treated MPTP-induced PD mice showed 84 differentially expressed genes (DEGs) and 1804 differentially methylated regions (DMRs). Upregulated genes involved zinc ion homeostasis, cilium protein localization, and transcription; downregulated genes linked to ephrin receptor signaling, somitogenesis, and gene expression regulation. Hyper/hypomethylated DMRs post-ARE treatment associated with Wnt signaling, mitochondrial organization, dopamine biosynthesis, and hindbrain development. No significant correlation between DEGs and methylated genes related to PD pathogenesis. This research has identified the epigenetic targets of ARE's therapeutic action and gives insight on how ARE protects neurons in Parkinson's disease.

Studying Mitotic Phosphorylation in Drosophila.

Mitosis is largely controlled by the reversible phosphorylation of effector proteins. The addition or removal of phosphate groups alters the activities of these proteins, resulting in changes in chromosome structure, cytoskeletal dynamics, nuclear envelope integrity, and other transformations that must occur as a cell progresses through mitosis. Drosophila has been instrumental in the elucidation of the molecular mechanisms of mitosis, which are mostly conserved among animals. In this model system, sophisticated genetic tools can be used to study mitosis in different tissues during development in vivo. Drosophila cell culture affords complementary possibilities. In this chapter, we present a phosphoproteomic protocol using Drosophila cell culture to identify phosphorylation sites that depend on mitotic kinases and phosphatases. We also provide protocols to dissect the roles of the identified sites in the regulation of protein interactions and localization during mitosis, using Drosophila embryos. We emphasize the advantages of the selected methods compared to possible alternatives in Drosophila or in other systems.

Andrographolide attenuates SARS-CoV-2 infection via an up-regulation of glutamate-cysteine ligase catalytic subunit (GCLC)

BackgroundAndrographolide is a medicinal compound which possesses anti-SARS-CoV-2 activity. A number of cellular targets of andrographolide have been identified by target predictions and computational studies. PurposeHowever, a potential cellular target of andrographolide has never been explored in SARS-CoV-2 infected lung epithelial cells. We aimed to identify cellular pathways involved in andrographolide-mediated anti-SARS-CoV-2 activity. MethodsThe viral infection was determined by immunofluorescence staining, enzyme-linked immunosorbent assay and focus-forming assay. Proteomic analysis was employed to identify cellular pathways and key proteins controlled by andrographolide in the human lung epithelial cells Calu-3 infected by SARS-CoV-2. Immunofluorescence staining was used to test protein expression and localization. Western blot and realtime PCR were utilized to elucidate gene expression. Cellular glutathione level was examined by a reduced/oxidized glutathione assay. An ectopic gene expression was delivered by plasmid transfection. ResultsGene ontology analysis indicates that proteins involved in nuclear factor erythroid 2-related factor 2 (NRF2)-regulated pathways were differentially expressed by andrographolide. Notably, andrographolide increased expression and nuclear localization of the transcription factor NRF2. In addition, transcriptional expression of GCLC and glutamate-cysteine ligase modifier subunit (GCLM), which are NRF2 target genes, were induced by andrographolide. We further find that infection of SARS-CoV-2 resulted in a reduction of glutathione level in Calu-3; the effect that was rescued by andrographolide. Moreover, andrographolide also induced expression of the glutathione producing enzyme GCLC in SARS-CoV-2 infected lung epithelial cells. Importantly, an ectopic over-expression of GCLC or treatment of N-acetyl-L-cysteine in Calu-3 cells led to a decrease in SARS-CoV-2 infection. ConclusionCollectively, our findings suggest the interplay between GCLC-mediated glutathione biogenesis induced by andrographolide and the anti-SARS-CoV-2 activity. The glutathione biogenesis and recycling pathways should be further exploited as a targeted therapy against SARS-CoV-2 infection.

The protein composition of human adenovirus replication compartments.

Human adenoviruses are double-stranded DNA viruses that replicate in the cell nucleus and induce the formation of replication compartments (RCs) that are critical in viral replication and control of virus-host interactions. RCs are specialized virus-induced subnuclear microenvironments where not only viral genome replication and expression are orchestrated but also host proteins that restrict viral replication are co-opted and subverted. The protein composition of these RCs remains largely unexplored. In this study, we isolated adenovirus RC-enriched fractions from infected cells at different times post-infection and employed a tandem mass tag-based quantitative mass spectrometry approach to identify proteins associated with RCs (data available via ProteomeXchange identifier PXD051745). These findings reveal an elaborate network of host and viral proteins potentially relevant for RC formation and function. To validate the RC-protein components identified by mass spectrometry, we employed immunofluorescence and immunoblotting techniques. Proteins previously described to colocalize in RCs in infected cells were identified in the isolated subnuclear fractions. In addition, we validated newly identified proteins associated with RCs, including the high mobility group box 1 (HMGB1), the SET nuclear proto-oncogene, the structure-specific recognition protein 1 (SSRP1), the CCCTC-binding protein (CTCF), and sirtuin 6 (SIRT6). We identified HMGB1 as a protein that binds to the viral DNA binding protein (DBP). Using shRNA knockdowns and inhibitors, we demonstrated that HMGB1 acts as a proviral factor, promoting efficient viral DNA synthesis and progeny production. Our data further suggest potential candidate targets for therapeutic intervention and provide mechanistic insights into the molecular basis of virus-host interactions.IMPORTANCEHuman adenoviruses serve as models for studying respiratory viruses and have provided critical insights into viral genome replication and gene expression, as well as the control of virus-host interactions. These processes are coordinated within virus-induced subnuclear microenvironments known as RCs. We conducted quantitative proteome analyses of RC-enriched subnuclear fractions at different times post-infection with human adenovirus species C type 5, revealing a multifaceted network of proteins that participate in the regulation of gene expression, DNA damage response, RNA metabolism, innate immunity, and other cellular antiviral defense mechanisms. Furthermore, we validated the localization of several host proteins to viral RCs using immunofluorescence microscopy and immunoblotting and identified cellular HMGB1 as a proviral factor late during infection. These findings represent the first analysis of the proteomes of isolated RCs and not only enhance our understanding of nuclear organization during infection but also shed light on the complex interplay between viral and host factors within RCs.

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.