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Background: The activation of the DNA damage response (DDR) heavily relies on post-translational modifications (PTMs) of proteins, which play a crucial role in the prevention of genetic instability and tumorigenesis. Among these PTMs, palmitoylation is a highly conserved process that is dysregulated in numerous cancer types. However, its direct involvement in the DDR and the underlying mechanisms remain unclear. Methods: CRISPR-Cas9 technology was used to generate the PORCN KO and PORCN NLS KO cell lines. The effects of PORCN NLS in the DDR were verified by colony formation assays, MTT assays, the DR/EJ5 homologous recombination/non-homologous end-joining reporter system, xenograft tumor growth and immunofluorescence. Mechanisms were explored by mass spectrometry, acyl-biotin exchange (ABE) palmitoylation assay, Click-iT assay, cell subcellular fractionation assay, Western blot analysis, and in vivo and in vitro co-immunoprecipitation. Results: In this study, we introduce evidence that Porcupine (PORCN) is an integral component of and plays a critical role in the DDR. PORCN deficiency hampers nonhomologous end joining (NHEJ) and highly sensitizes cells to ionizing radiation (IR) both in vitro and in vivo. We also provide evidence that PORCN possesses a nuclear fraction (nPORCN) with S-acyltransferase activity, unlike its membrane-bound O-acyltransferase in the endoplasmic reticulum. Furthermore, we show that nPORCN is necessary for the successful activation of NHEJ. Using mass spectrometry, we reveal the existence of an nPORCN complex and show that nPORCN mediates the S-palmitoylation of XRCC6/Ku70 at five specific cysteine sites in response to IR. Mutation of these sites causes a substantial increase in radiosensitivity and delays NHEJ. Additionally, we present evidence that nPORCN-dependent Ku70 palmitoylation is required for DNA-PKcs/Ku70/Ku80 complex formation. Conclusion: Our findings underscore the crucial role of nPORCN-dependent Ku70 S-palmitoylation in the DDR. [ABSTRACT FROM AUTHOR]

C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors. [ABSTRACT FROM AUTHOR]

The article discusses a retracted study on the potential of Santacruzamate A (STA) in treating Alzheimer's disease (AD) by targeting endoplasmic reticulum (ER) stress pathways. The study found that STA could alleviate Aβ-induced toxicity in cells and improve cognitive deficits in mice by modulating ER stress and apoptotic pathways. By inhibiting unfolded protein response (UPR) and ER stress, STA showed promise in treating AD by regulating ER retention signal receptors and mitochondrial assembly proteins. The findings suggest that STA, a carbamate derivative, may hold potential in treating neurodegenerative disorders by targeting ER stress pathways. [Extracted from the article]

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance. Author summary: Bacterial conjugation plays a pivotal role in the evolution of bacterial populations. The circulation of drug resistance genes bolsters the emergence of multidrug-resistant pathogens, with which contemporary medicine struggles to cope. Exclusion is a natural process preventing the redundant acquisition of a plasmid via conjugation by a host harbouring an identical or similar plasmid. Although exclusion has been known for the past half-century, the mechanisms involved remain poorly understood. This study describes an exclusion factor, Sfx, encoded by IncC, IncA and related conjugative plasmids and by unrelated integrative and mobilizable elements. We report that Sfx is a lipoprotein of the recipient that selectively inhibits conjugation based on the adhesin TraN expressed at the surface of the donor. We propose a mechanistic model for Sfx-mediated exclusion. Ultimately, a better understanding of exclusion could facilitate the design of conjugation inhibitors targeting mating pair formation to curb the circulation of drug-resistance genes in healthcare settings, agriculture, animal husbandry and food and drug production. [ABSTRACT FROM AUTHOR]

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway. [ABSTRACT FROM AUTHOR]

Spatial regulation of RNA plays a critical role in gene expression regulation and cellular function. Understanding spatially resolved RNA dynamics and translation is vital for bringing new insights into biological processes such as embryonic development, neurobiology, and disease pathology. This review explores past studies in subcellular, cellular, and tissue-level spatial RNA biology driven by diverse methodologies, ranging from cell fractionation, in situ and proximity labeling, imaging, spatially indexed next-generation sequencing (NGS) approaches, and spatially informed computational modeling. Particularly, recent advances have been made for near-genome-scale profiling of RNA and multimodal biomolecules at high spatial resolution. These methods enabled new discoveries into RNA's spatiotemporal kinetics, RNA processing, translation status, and RNA-protein interactions in cells and tissues. The evolving landscape of experimental and computational strategies reveals the complexity and heterogeneity of spatial RNA biology with subcellular resolution, heralding new avenues for RNA biology research. Spatiotemporal RNA regulation is critical for executing and fine-tuning RNA function and protein synthesis in cells. Exploring the cutting-edge methodologies, including spatial omics techniques, Ren et al. highlight recent advances enabling the comprehensive profiling of RNA distribution and dynamics, paving the way for innovative RNA research. [ABSTRACT FROM AUTHOR]

A previous study using Thermostable Group II Intron Reverse Transcriptase sequencing (TGIRT-seq) found human plasma contains short (≤300 nt) structured full-length excised linear intron (FLEXI) RNAs with potential to serve as blood-based biomarkers. Here, TGIRT-seq identified >9,000 different FLEXI RNAs in human cell lines, including relatively abundant FLEXIs with cell-type-specific expression patterns. Analysis of public CLIP-seq datasets identified 126 RNA-binding proteins (RBPs) that have binding sites within the region corresponding to the FLEXI or overlapping FLEXI splice sites in pre-mRNAs, including 53 RBPs with binding sites for ≥30 different FLEXIs. These included splicing factors, transcription factors, a chromatin remodeling protein, cellular growth regulators, and proteins with cytoplasmic functions. Analysis of ENCODE datasets identified subsets of these RBPs whose knockdown impacted FLEXI host gene mRNA levels or proximate alternative splicing, indicating functional interactions. Hierarchical clustering identified six subsets of RBPs whose FLEXI binding sites were co-enriched in six subsets of functionally related host genes: AGO1-4 and DICER, including but not limited to agotrons or mirtron pre-miRNAs; DKC1, NOLC1, SMNDC1, and AATF (Apoptosis Antagonizing Transcription Factor), including but not limited to snoRNA-encoding FLEXIs; two subsets of alternative splicing factors; and two subsets that included RBPs with cytoplasmic functions (e.g., LARP4, PABPC4, METAP2, and ZNF622) together with regulatory proteins. Cell fractionation experiments showed cytoplasmic enrichment of FLEXI RNAs with binding sites for RBPs with cytoplasmic functions. The subsets of host genes encoding FLEXIs with binding sites for different subsets of RBPs were co-enriched with non-FLEXI other short and long introns with binding sites for the same RBPs, suggesting overarching mechanisms for coordinately regulating expression of functionally related genes. Our findings identify FLEXIs as a previously unrecognized large class of cellular RNAs and provide a comprehensive roadmap for further analyzing their biological functions and the relationship of their RBPs to cellular regulatory mechanisms. Author summary: We characterized a previously unrecognized, large class of full-length excised liner intron (FLEXI) RNAs that are ≤300 nucleotides in length and relatively stable in human cells and plasma. These included subsets of FLEXIs with binding sites for different subsets of RNA-binding proteins (RBPs) that have different cellular functions and are co-enriched in subsets of functionally related genes with other short and long introns with binding sites for the same RBPs, potentially enabling coordinate regulation of different gene sets. Many of the proteins that have binding sites in different subsets of FLEXIs were also identified as interacting with each other by STRING protein-network analysis, potentially enabling co-localization of subsets of proteins to different intracellular locations, including the cytoplasm. Our findings provide a comprehensive road map for further analysis of the functions of FLEXIs and their associated RBPs and for the identification of FLEXIs that can serve as biomarkers in human cells and plasma. [ABSTRACT FROM AUTHOR]

Numerous Treponema species are prevalent in the dysbiotic subgingival microbial community during periodontitis. The major outer sheath protein is a highly expressed virulence factor of the well‐characterized species Treponema denticola. Msp forms an oligomeric membrane protein complex with adhesin and porin properties and contributes to host–microbial interaction. Treponema maltophilum and Treponema lecithinolyticum species are also prominent during periodontitis but are relatively understudied. Msp‐like membrane surface proteins exist in T. maltophilum (MspA) and T. lecithinolyticum (MspTL), but limited information exists regarding their structural features or functionality. Protein profiling reveals numerous differences between these species, but minimal differences between strains of the same species. Using protein modeling tools, we predict MspA and MspTL monomeric forms to be large β‐barrel structures composed of 20 all‐next‐neighbor antiparallel β strands which most likely adopt a homotrimer formation. Using cell fractionation, Triton X‐114 phase partitioning, heat modifiability, and chemical and detergent release assays, we found evidence of amphiphilic integral membrane‐associated oligomerization for both native MspA and MspTL in intact spirochetes. Proteinase K accessibility and immunofluorescence assays demonstrate surface exposure of MspA and MspTL. Functionally, purified recombinant MspA or MspTL monomer proteins can impair neutrophil chemotaxis. Expressions of MspA or MspTL with a PelB leader sequence in Escherichia coli also demonstrate surface exposure and can impair neutrophil chemotaxis in an in vivo air pouch model of inflammation. Collectively, our data demonstrate that MspA and MspTL membrane proteins can contribute to pathogenesis of these understudied oral spirochete species. [ABSTRACT FROM AUTHOR]

OsS1Fa1, a homologue of spinach S1Fa, is a small protein in rice that contains four distinct conserved motifs and participates in drought tolerance. However, the biological functions of these conserved motifs have not been characterized to date. Therefore, we investigated the roles of these conserved domains in the localization and cellular function of OsS1Fa1. We analysed the subcellular localization of OsS1Fa1 using confocal laser scanning microscopy (CLSM), following particle bombardment and bacterial infiltration. An E. coli in vivo reconstituted sumoylation assay was conducted to investigate sumoylation of OsS1Fa1. We characterized the function of the transmembrane domain of OsS1Fa1 in drought tolerance using transgenic Arabidopsis plants. Fluorescence analysis showed that OsS1Fa1 localized to the nuclear and cytoplasmic membranes. Mutation and cell fractionation analyses revealed that the membrane localization domain determined the subcellular localization of OsS1Fa1. The rice homologue OsS1Fa2 and Arabidopsis orthologs AtS1Fa1, AtS1Fa2, and AtS1Fa3 also exhibited similar localization patterns as OsS1Fa1. Sumoylation analysis demonstrated that OsS1Fa1 was conjugated with the small ubiquitin‐related modifier (SUMO). Transgenic analysis showed that overexpression of OsS1Fa1(TMm1), a mutant form of the transmembrane domain of OsS1Fa1, in Arabidopsis did not enhance drought stress tolerance, whereas OsS1Fa1 overexpression improved the drought tolerance of transgenic Arabidopsis. Our data indicate that rice and Arabidopsis S1Fa1 proteins localize in the nuclear and cytoplasmic membranes, and that transmembrane domain determines subcellular localization and plays an important role in drought stress tolerance. [ABSTRACT FROM AUTHOR]

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain. [ABSTRACT FROM AUTHOR]

Summary: Transcription and export (TREX) is a multi‐subunit complex that links synthesis, processing and export of mRNAs. It interacts with the RNA helicase UAP56 and export factors such as MOS11 and ALYs to facilitate nucleocytosolic transport of mRNAs. Plant MOS11 is a conserved, but sparsely researched RNA‐binding export factor, related to yeast Tho1 and mammalian CIP29/SARNP.Using biochemical approaches, the domains of Arabidopsis thaliana MOS11 required for interaction with UAP56 and RNA‐binding were identified. Further analyses revealed marked genetic interactions between MOS11 and ALY genes. Cell fractionation in combination with transcript profiling demonstrated that MOS11 is required for export of a subset of mRNAs that are shorter and more GC‐rich than MOS11‐independent transcripts.The central α‐helical domain of MOS11 proved essential for physical interaction with UAP56 and for RNA‐binding. MOS11 is involved in the nucleocytosolic transport of mRNAs that are upregulated under stress conditions and accordingly mos11 mutant plants turned out to be sensitive to elevated NaCl concentrations and heat stress.Collectively, our analyses identify functional interaction domains of MOS11. In addition, the results establish that mRNA export is critically involved in the plant response to stress conditions and that MOS11 plays a prominent role at this. [ABSTRACT FROM AUTHOR]

The pretreatment of lignocellulosic biomass such as Miscanthus grown on marginal agricultural land is very challenging and requires severe conditions to fractionate cell wall polymers for further valorization. The current study aimed to determine organic acid-based mild conditions to pretreat contrasting lignocellulosic Miscanthus genotypes for the efficient fractionation of cell wall components, with special focus on hemicellulose extraction. In doing so, five Miscanthus genotypes were subjected to four different acid treatments (sulfuric acid, oxalic acid, malonic acid, and citric acid) in a vertical high-pressure steam sterilizer. The results demonstrated that, among the organic acids, oxalic acid was identified as the most effective pretreatment solvent for hemicellulose separation, whereas citric acid yielded the highest amount of galacturonic acid, varying from 15 to 17 mg mL−1 across genotypes. One best performing genotype was selected for the enzymatic hydrolysis. Overall, M. floridulus genotypes exhibited the optimal quality traits for efficient bioconversion with second best in terms of ethanol production potential. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are a population of innate immune cells that have cytotoxic functions and are involved in protecting the body from viruses and transformed cells. Placental development is largely determined by the interaction of decidual NK cells and trophoblast cells. During pregnancy, NK cells accumulate around trophoblast cells, while regulating trophoblast proliferation, migration and invasion through the secretion of cytokines and growth factors. The trophoblast, in turn, secretes chemokines and expresses ligands for NK cell adhesion receptors. Thus, functional regulation of trophoblast and NK cells is reciprocal. Despite intensive research, the role of NK cells and methods for correcting their functional activity in reproduction remain controversial. The aim of this study was to assess the effect of NK cell lysate protein fractions on the phenotype, migration and apoptosis of trophoblast cells in an in vitro model experiment, using a new methodological approach. Chromatographic separation yielded six fractions with different protein cargoes. It was found that CD105 (endoglin) expression on the surface of JEG-3 trophoblast cells after their cultivation in the presence of high- (< 250 kDa) or low-molecular-weight (< 45 kDa) fractions of NK-92 whole-cell lysate was reduced compared to spontaneous expression, with the relative count of trophoblast cells with a CD105+ phenotype being also lowered. In addition, one of the low-molecular-weight fractions decreased TRAIL-R2 receptor expression by trophoblast cells. The high-molecular-weight fractions did not enable trophoblast cells to migrate completely through a semi-permeable membrane, with the area occupied by the migrated cells not exceeding the baseline control area. Moreover, the high-molecular-weight fraction containing the TGFβ dimer increased p-SMAD2/3 level in trophoblast cells 1 h after co-culture, followed by a decrease in the level of this phosphorylated form after 2 or more hours, and also elevated procaspase-3 level one day after co-culture. The data obtained hypothetically reflect the possible behavior of chorion cells under the influence of collapsing NK cells in the event of their death under both normal and pathological conditions caused by viral and bacterial infections, as well as other stress factors leading to reproductive pathology. [ABSTRACT FROM AUTHOR]

A novel millifluidic process introduces age-based fractionation of S. pastorianus var. carlsbergensis yeast culture through magnetophoresis. Saccharomyces yeast is a model organism for aging research used in various industries. Traditional age-based cell separation methods were labor-intensive, but techniques like magnetic labeling have eased the process by being non-invasive and scalable. Our approach introduces an age-specific fractionation using a 3D-printed millfluidic chip in a two-step process, ensuring efficient cell deflection in the magnetic field and counteracting magnetic induced convection. Among various channel designs, the pinchshaped channel proved most effective for age differentiation based on magnetically labeled bud scar numbers. Metabolomic analyses revealed changes in certain amino acids and increased NAD+ levels, suggesting metabolic shifts in aging cells. Gene expression studies further underlined these age-related metabolic changes. This innovative platform offers a high-throughput, non-invasive method for age-specific yeast cell fractionation, with potential applications in industries ranging from food and beverages to pharmaceuticals. [ABSTRACT FROM AUTHOR]

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase), the ratelimiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examined its lipid dropletlocalized pool using complementary immunofluorescence and cell fractionation approaches and found that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of truncated SM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis. [ABSTRACT FROM AUTHOR]

Background: Small nucleolar RNAs (snoRNAs) play key roles in ribosome biosynthesis. However, the mechanism by which snoRNAs regulate cancer stemness remains to be fully elucidated. Methods: SNORA68 expression was evaluated in breast cancer tissues by in situ hybridization and qRT‒PCR. Proliferation, migration, apoptosis and stemness analyses were used to determine the role of SNORA68 in carcinogenesis and stemness maintenance. Mechanistically, RNA pull-down, RNA immunoprecipitation (RIP), cell fractionation and coimmunoprecipitation assays were conducted. Results: SNORA68 exhibited high expression in triple-negative breast cancer (TNBC) and was significantly correlated with tumor size (P = 0.048), ki-67 level (P = 0.037), and TNM stage (P = 0.015). The plasma SNORA68 concentration was significantly lower in patients who achieved clinical benefit. The SNORA68-high patients had significantly shorter disease-free survival (DFS) (P = 0.036). Functionally, SNORA68 was found to promote the cell stemness and carcinogenesis of TNBC in vitro and in vivo. Furthermore, elevated SNORA68 expression led to increased nucleolar RPL23 expression and retained RPL23 in the nucleolus by binding U2AF2. RPL23 in the nucleolus subsequently upregulated c-Myc expression. This pathway was validated using a xenograft model. Conclusion: U2AF2-SNORA68 promotes TNBC stemness by retaining RPL23 in the nucleolus and increasing c-Myc expression, which provides new insight into the regulatory mechanism of stemness. [ABSTRACT FROM AUTHOR]

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and μ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with μ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four μ-subunits (μ1, μ2, μ3A and μ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with μ3A was more robust than that with other μ-subunits. LAMP-2B-CT interacted exclusively and moderately with μ3A. LAMP-2C-CT did not detectably interact with any of the four μ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3. [ABSTRACT FROM AUTHOR]

Conformational diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington’s disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.

Background: Pancreatic ductal adenocarcinoma is an aggressive cancer type with one of the lowest survival rates due to late diagnosis and the absence of effective treatments. A better understanding of PDAC biology will help researchers to discover the Achilles' heel of cancer cells. In that regard, our research team investigated the function of an emerging oncoprotein known as myoferlin. Myoferlin is overexpressed in PDAC and its silencing/targeting has been shown to affect cancer cell proliferation, migration, mitochondrial dynamics and metabolism. Nevertheless, our comprehension of myoferlin functions in cells remains limited. In this study, we aimed to understand the molecular mechanism linking myoferlin silencing to mitochondrial dynamics. Methods: Experiments were performed on two pancreas cancer cell lines, Panc-1 and MiaPaCa-2. Myoferlin localization on mitochondria was evaluated by immunofluorescence, proximity ligation assay, and cell fractionation. The presence of myoferlin in mitochondria-associated membranes was assessed by cell fractionation and its function in mitochondrial calcium transfer was evaluated using calcium flow experiments, proximity ligation assays, co-immunoprecipitation, and timelapse fluorescence microscopy in living cells. Results: Myoferlin localization on mitochondria was investigated. Our results suggest that myoferlin is unlikely to be located on mitochondria. Instead, we identified myoferlin as a new component of mitochondria-associated membranes. Its silencing significantly reduces the mitochondrial calcium level upon stimulation, probably through myoferlin interaction with the inositol 1,4,5-triphosphate receptors 3. Conclusions: For the first time, myoferlin was specifically demonstrated to be located in mitochondria-associated membranes where it participates to calcium flow. We hypothesized that this function explains our previous results on mitochondrial dynamics. This study improves our comprehension of myoferlin localization and function in cancer biology. [ABSTRACT FROM AUTHOR]

The ubiquitin/26S proteasome system is a crucial regulatory mechanism that governs various cellular processes in plants, including signal transduction, transcriptional regulation, and responses to biotic and abiotic stressors. Our study shows that the RING-H2-type E3 ubiquitin ligase, Arabidopsis Tóxicos en Levadura 2 (ATL2), is involved in response to fungal pathogen infection. Under normal growth conditions, the expression of the ATL2 gene is low, but it is rapidly and significantly induced by exogenous chitin. Additionally, ATL2 protein stability is markedly increased via chitin treatment, and its degradation is prolonged when 26S proteasomal function is inhibited. We found that an atl2 null mutant exhibited higher susceptibility to Alternaria brassicicola, while plants overexpressing ATL2 displayed increased resistance. We also observed that the hyphae of A. brassicicola were strongly stained with trypan blue staining, and the expression of A. brassicicola Cutinase A (AbCutA) was dramatically increased in atl2. In contrast, the hyphae were weakly stained, and AbCutA expression was significantly reduced in ATL2-overexpressing plants. Using bioinformatics, live-cell confocal imaging, and cell fractionation analysis, we revealed that ATL2 is localized to the plasma membrane. Further, it is demonstrated that the ATL2 protein possesses E3 ubiquitin ligase activity and found that cysteine 138 residue is critical for its function. Moreover, ATL2 is necessary to successfully defend against the A. brassicicola fungal pathogen. Altogether, our data suggest that ATL2 is a plasma membrane-integrated protein with RING-H2-type E3 ubiquitin ligase activity and is essential for the defense response against fungal pathogens in Arabidopsis. [ABSTRACT FROM AUTHOR]

Beer yeast is an excellent source of β-glucans or mono-and oligosaccharides, which have various biological and technical functional properties and can be used as food ingredients. The steps involved in extracting β-glucan from beer yeast cells, such as cell wall disruption and purification, have notable effects on the compound's yield, cost, and various properties, including biological, physicochemical, and technical aspects. In this study, by comparing the extraction efficiencies of 6 synthetic deep eutectic solvents (DESs) for β-glucan in brewer's yeast, it was found that using DES composed of choline chloride and lactic acid resulting in the yield and purity of insoluble β-glucan (IBG) was 1.30 g/g and 49.3%. Even after one cycle of DES, IBG could still be separated with a yield of 0.26 g/g and a purity of 39.1%. Additionaly, the molecular weight of IBG separated by the DES method was 1.0–1.6 kDa, significantly lower than the range obtained using the traditional alkali-alcohol separation method (39.8–656 kDa). The results suggest an alternative use of DES for spent beer yeast byproduct extraction. [Display omitted] • Six deep eutectic solvents (DESs) used in brewer's yeast valorization. • Using a CLA (choline chloride and lactic acid) DES results in high yield and purity of β-glucan. • β-glucan with a yield 0.26 g/g and purity 39.1% after a single recycle of DES [ABSTRACT FROM AUTHOR]

The importance of lung microbiome changes in developing chronic lung allograft dysfunction (CLAD) after lung transplantation is poorly understood. The lung microbiome–immune interaction may be critical in developing CLAD. In this context, examining alterations in the microbiome and immune cells of the lungs following CLAD, in comparison to the lung condition immediately after transplantation, can offer valuable insights. Four adult patients who underwent lung retransplantation between January 2019 and June 2020 were included in this study. Lung tissues were collected from the same four individuals at two different time points: at the time of the first transplant and at the time of the explantation of CLAD lungs at retransplantation due to CLAD. We analyzed whole-genome sequencing using the Kraken2 algorithm and quantified the cell fractionation from the bulk tissue gene expression profile for each lung tissue. Finally, we compared the differences in lung microbiome and immune cells between the lung tissues of these two time points. The median age of the recipients was 57 years, and most (75%) had undergone lung transplants for idiopathic pulmonary fibrosis. All patients were administered basiliximab for induction therapy and were maintained on three immunosuppressants. The median CLAD-free survival term was 693.5 days, and the median time to redo the lung transplant was 843.5 days. Bacterial diversity was significantly lower in the CLAD lungs than at transplantation. Bacterial diversity tended to decrease according to the severity of the CLAD. Aerococcus, Caldiericum, Croceibacter, Leptolyngbya, and Pulveribacter genera were uniquely identified in CLAD, whereas no taxa were identified in lungs at transplantation. In particular, six taxa, including Croceibacter atlanticus, Caldiserium exile, Dolichospermum compactum, Stappia sp. ES.058, Kinetoplastibacterium sorsogonicusi, and Pulveribacter suum were uniquely detected in CLAD. Among immune cells, CD8+ T cells were significantly increased, while neutrophils were decreased in the CLAD lung. In conclusion, unique changes in lung microbiome and immune cell composition were confirmed in lung tissue after CLAD compared to at transplantation. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a delicate and complex biological process in which spermatogonial stem cells continue to proliferate and differentiate into mature spermatozoa, maintaining sperm production in male mammals throughout the lifetime. To study the molecular mechanism of spermatogenesis, researchers had to isolate different germ cell subpopulations for in vitro culture and characterization. However, due to the existence of several stages of germ cells and a variety of populations of somatic cells in the testis of male mammals, it is a challenge for us to obtain high-purity germ cell subpopulations for further research. Here, we optimized the STA-PUT device and successfully applied it to isolate and purify spermatogonia populations in piglets, and multiple germ cell populations at different developmental stages in testes of adult mice and boars. This work provides a simple platform for germ cell fractionation to facilitate the molecular mechanistic study of animal spermatogenesis in vitro. • We established an optimized velocity sedimentation STA-PUT device for cell fractionation. • The STA-PUT device was applied to isolate multiple germ cell populations in mice and pigs. • The STA-PUT device can isolate spermatogonia, spermatocytes, and spermatids in a high purity and a sufficient number. [ABSTRACT FROM AUTHOR]

Purpose: Enhancing chemotherapy sensitivity in colorectal cancer (CRC) is critical for improving treatment outcomes. TMEM120A has been reported to interact with coenzyme A (CoA), but its biological significance in CRC is unknown. In this study, we aimed to investigate the functional implications of TMEM120A in CRC and its impact on chemotherapy sensitivity. Methods: Stable knockout of TMEM120A in CRC cell lines was conducted using CRISPR/Cas9 technology. Overexpression of various derivatives of TMEM120A was achieved through lentiviral transduction. Cell fractionation was employed to isolate the nuclear and cytoplasmic fraction. Total histones were isolated by acid extraction and then subjected to determine histone acetylation levels using western blot analysis. Cell viability was evaluated using the MTS assay. Results: We demonstrate that TMEM120A's nuclear localization is crucial for its role in regulating CRC chemosensitivity. Mechanistically, the nuclear subpopulation of TMEM120A plays a key role in sustaining the nuclear CoA levels, which in turn influences the levels of nuclear acetyl-CoA and histone acetylation in CRC cells. Notably, direct inhibition of histone acetylation recapitulated the phenotypic effects observed upon TMEM120A depletion, leading to increased chemosensitivity in CRC cells. Conclusion: Our study provides novel insights into the role of TMEM120A in modulating chemotherapy sensitivity in CRC. Nuclear TMEM120A regulates CoA levels, which in turn modulates nuclear acetyl-CoA levels and histone acetylation, thereby influencing the response of CRC cells to chemotherapy agents. Targeting TMEM120A-mediated pathways may represent a promising strategy for enhancing chemotherapy efficacy in CRC treatment. [ABSTRACT FROM AUTHOR]

Plant viruses depend on host cellular factors for their replication and movement. There are cellular proteins that change their localization and/or expression and have a proviral role or antiviral activity and interact with or target viral proteins. Identification of those proteins and their roles during infection is crucial for understanding plant–virus interactions and to design antiviral resistance in crops. Important host proteins have been identified using approaches such as tag-dependent immunoprecipitation or yeast two hybridization that require cloning individual proteins or the entire virus. However, the number of possible interactions between host and viral proteins is immense. Therefore, an alternative method is needed for proteome-wide identification of host proteins involved in host–virus interactions. Here, we present cell fractionation coupled with mass spectrometry as an option to identify protein–protein interactions between viruses and their hosts. This approach involves separating subcellular organelles using differential and/or gradient centrifugation from virus-free and virus-infected cells (1) followed by comparative analysis of the proteomic profiles obtained for each subcellular organelle via mass spectrometry (2). After biological validation, prospect host proteins with proviral or antiviral roles can be subject to fundamental studies in the context of basic biology to shed light on both virus replication and cellular processes. They can also be targeted via gene editing to develop virus-resistant crops. [ABSTRACT FROM AUTHOR]

Background: DNA damage response (DDR) and repair are vital for safeguarding genetic information and ensuring the survival and accurate transmission of genetic material. DNA damage, such as DNA double-strand breaks (DSBs), triggers a response where sensor proteins recognize DSBs. Information is transmitted to kinases, initiating a sequence resulting in the activation of the DNA damage response and recruitment of other DDR and repair proteins to the DSB site in a highly orderly sequence. Research has traditionally focused on individual protein functions and their order, with limited quantitative analysis, prompting this study's attempt at absolute quantification of DNA damage response and repair proteins and capturing changes in protein chromatin affinity after DNA damage through biochemical fractionation methods. Results: To assess the intracellular levels of proteins involved in DDR and repair, multiple proteins associated with different functions were quantified in EPC2-hTERT cells. H2AX had the highest intracellular abundance (1.93 × 106 molecules/cell). The components of the MRN complex were present at the comparable levels: 6.89 × 104 (MRE11), 2.17 × 104 (RAD50), and 2.35 × 104 (NBS1) molecules/cell. MDC1 was present at 1.27 × 104 molecules/cell. The intracellular levels of ATM and ATR kinases were relatively low: 555 and 4860 molecules/cell, respectively. The levels of cellular proteins involved in NHEJ (53BP1: 3.03 × 104; XRCC5: 2.62 × 104; XRCC6: 5.05 × 105 molecules/cell) were more than an order of magnitude higher than that involved in HR (RAD51: 2500 molecules/cell). Furthermore, we analyzed the dynamics of MDC1 and γH2AX proteins in response to DNA damage induced by the unstable agent neocarzinostatin (NCS). Using cell biochemical fractionation, cells were collected and analyzed at different time points after NCS exposure. Results showed that γH2AX in chromatin fraction peaked at 1 h post-exposure and gradually decreased, while MDC1 translocated from the isotonic to the hypertonic fraction, peaking at 1 hour as well. The study suggests increased MDC1 affinity for chromatin through binding to γH2AX induced by DNA damage. The γH2AX-bound MDC1 (in the hypertonic fraction) to γH2AX ratio at 1 h post-exposure was 1:56.4, with lower MDC1 levels which may attributed to competition with other proteins. Conclusions: The approach provided quantitative insights into protein dynamics in DNA damage response. [ABSTRACT FROM AUTHOR]

Mitochondrial transplantation is emerging as a novel cellular biotherapy to alleviate mitochondrial damage and dysfunction. Mitochondria play a crucial role in establishing cellular homeostasis and providing cell with the energy necessary to accomplish its function. Owing to its endosymbiotic origin, mitochondria share many features with their bacterial ancestors. Unlike the nuclear DNA, which is packaged into nucleosomes and protected from adverse environmental effects, mitochondrial DNA are more prone to harsh environmental effects, in particular that of the reactive oxygen species. Mitochondrial damage and dysfunction are implicated in many diseases ranging from metabolic diseases to cardiovascular and neurodegenerative diseases, among others. While it was once thought that transplantation of mitochondria would not be possible due to their semiautonomous nature and reliance on the nucleus, recent advances have shown that it is possible to transplant viable functional intact mitochondria from autologous, allogenic, and xenogeneic sources into different cell types. Moreover, current research suggests that the transplantation could positively modulate bioenergetics and improve disease outcome. Mitochondrial transplantation techniques and consequences of transplantation in cardiomyocytes are the theme of this review. We outline the different mitochondrial isolation and transfer techniques. Finally, we detail the consequences of mitochondrial transplantation in the cardiovascular system, more specifically in the context of cardiomyopathies and ischemia.

The inability to inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Previous work addressed this challenge by analyzing metabolism in isolated organelles, which grossly bias metabolic activity. Here, we describe a method for inferring physiological metabolic fluxes and metabolite concentrations in mitochondria and cytosol based on isotope tracing experiments performed with intact cells. This is made possible by computational deconvolution of metabolite isotopic labeling patterns and concentrations into cytosolic and mitochondrial counterparts, coupled with metabolic and thermodynamic modelling. Our approach lowers the uncertainty regarding compartmentalized fluxes and concentrations by one and three orders of magnitude compared to existing modelling approaches, respectively. We derive a quantitative view of mitochondrial and cytosolic metabolic activities in central carbon metabolism across cultured cell lines without performing cell fractionation, finding major variability in compartmentalized malate-aspartate shuttle fluxes. We expect our approach for inferring metabolism at a subcellular resolution to be instrumental for a variety of studies of metabolic dysfunction in human disease and for bioengineering. Studying metabolism in distinct subcellular compartments typically involves isolating organelles. Here, the authors demonstrate a quantitative approach to infer cytosolic and mitochondrial metabolic activities based on experiments with intact cells, maintaining physiological conditions. [ABSTRACT FROM AUTHOR]

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of themolecular role of the proteasewithin Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation byMTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation. [ABSTRACT FROM AUTHOR]

G1P3/IFI6 is an interferon stimulated gene with antiapoptotic, prometastatic, and antiviral functions. Despite its pleiotropic functions, subcellular localization of G1P3 remains unclear. Using biochemical‐ and confocal microscopic approaches, this study identified the localization of G1P3 in organelles of the endomembrane system and in the mitochondria of breast cancer cells. In cell fractionation studies, both interferon‐induced endogenous‐ and stably expressed G1P3 cofractionated with affinity‐isolated mitochondria. Results of the protease protection assay have suggested that ~24% of mitochondrial G1P3 resides within the mitochondria. Conforming to this, confocal microscopy studies of cells stably expressing epitope‐tagged G1P3 (MCF‐7/G1P3‐FLAG), identified its localization in mitochondria (~38%) as well as in ER, trans‐Golgi network (TGN), lysosomes, and in RAB5 positive (RAB5+) endosomes. These results suggested the trafficking of G1P3 from TGN into endolysosomes. Both G1P3 and RAB5 were known to confer apoptosis resistance through mitochondrial stabilization. Therefore, the effects of G1P3 on the localization of RAB5 in mitochondria were tested. Compared to vector control, the co‐occurrence of RAB5 with the mitochondria was increased by 1.5‐fold in MCF‐7/G1P3‐FLAG expressing cells (p ≤.005). Taken together, our results demonstrate a role for G1P3 to promote the association of RAB5+ endosomes with mitochondria and provide insight into yet another mechanism of G1P3‐induced cancer cell survival. [ABSTRACT FROM AUTHOR]

Zinc, an essential trace element that serves as a cofactor for numerous cellular and viral proteins, plays a central role in the dynamics of HIV-1 infection. Among the viral proteins, the nucleocapsid NCp7, which contains two zinc finger motifs, is abundantly present viral particles and plays a crucial role in coating HIV-1 genomic RNA, thus concentrating zinc within virions. In this study, we investigated whether HIV-1 virus production impacts cellular zinc homeostasis and whether isotopic fractionation occurs between the growth medium, the producing cells, and the viral particles. We found that HIV-1 captures a significant proportion of cellular zinc in the neo-produced particles. Furthermore, as cells grow, they accumulate lighter zinc isotopes from the medium, resulting in a concentration of heavier isotopes in the media, and the viruses exhibit a similar isotopic fractionation to the producing cells. Moreover, we generated HIV-1 particles in HEK293T cells enriched with each of the five zinc isotopes to assess the potential effects on the structure and infectivity of the viruses. As no strong difference was observed between the HIV-1 particles produced in the various conditions, we have demonstrated that enriched isotopes can be accurately used in future studies to trace the fate of zinc in cells infected by HIV-1 particles. Comprehending the mechanisms underlying zinc absorption by HIV-1 viral particles offers the potential to provide insights for developing future treatments aimed at addressing this specific facet of the virus's life cycle. [ABSTRACT FROM AUTHOR]

The Ca2+ release in microdomains formed by intercompartmental contacts, such as mitochondria-associated endoplasmic reticulum membranes (MAMs), encodes a signal that contributes to Ca2+ homeostasis and cell fate control. However, the composition and function of MAMs remain to be fully defined. Here, we focused on the transient receptor potential vanilloid 1 (TRPV1), a Ca2+-permeable ion channel and a polymodal nociceptor. We found TRPV1 channels in the reticular membrane, including some at MAMs, in a rat cardiomyoblast cell line (SV40-transformed H9c2) by Western blotting, immunostaining, cell fractionation, and proximity ligation assay. We used chemical and genetic probes to perform Ca2+ imaging in four cellular compartments: the endoplasmic reticulum (ER), cytoplasm, mitochondrial matrix, and mitochondrial surface. Our results showed that the ER Ca2+ released through TRPV1 channels is detected at the mitochondrial outer membrane and transferred to the mitochondria. Finally, we observed that prolonged TRPV1 modulation for 30 min alters the intracellular Ca2+ equilibrium and influences the MAM structure or the hypoxia/reoxygenation-induced cell death. Thus, our study provides the first evidence that TRPV1 channels contribute to MAM Ca2+ exchanges. [ABSTRACT FROM AUTHOR]

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool. Regulated subcellular localization changes control protein function. Here, the authors provide a seamless spatial proteomics pipeline for mapping whole-cell protein localization dynamics, which includes a scalable workflow and a software suite for automated data analysis and visualization. [ABSTRACT FROM AUTHOR]

Karl S. Matlin, Crossing the Boundaries of Life: Günter Blobel and the Origins of Molecular Cell Biology, Chicago: University of Chicago Press, 2022, ISBN: 9780226819235, 368 pp The last few years have seen a mini-boom of books on the history of modern molecular cell biology. While Blobel is Matlin's central ideas man, I Crossing the Boundaries of Life i is foremost a history of a scientific theory, along with the many investigative pathways that it joined together and the methods used to explicate it. [Extracted from the article]

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs. RNA localization is mediated by kinesin motors and anchoring. However, mechanisms underlying specificity are unclear. Here, the authors find that MBNL protein's zinc fingers prefer specific kinesins, and its unstructured tail mediates membrane anchoring. [ABSTRACT FROM AUTHOR]

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes. [ABSTRACT FROM AUTHOR]

Viral infection induces diverse cellular immune responses. Some viruses induce the production of antiviral cytokines, alterations of endogenous gene expression, and apoptosis; however, other viruses replicate without inducing such responses, enabling them to persistently infect cells. Infection by Borna disease virus type 1 (BoDV-1) can result in fatal immune-mediated encephalitis, including in humans, yet infection of cells in vitro is generally persistent. The regulatory mechanisms underlying this persistent infection remain unclear. Here, we show that an enhancer of RNA-silencing, TRBP, positively regulates BoDV RNA level in human cells. Knockdown of TRBP decreased BoDV RNA levels in persistently-infected cells, whereas overexpression of TRBP increased BoDV RNA levels. To investigate the mechanism underlying this phenomenon, we performed immunoprecipitation assays and found that TRBP interacts with BoDV RNA. Furthermore, we performed cell fractionation, which revealed that persistent infection with BoDV does not alter the localization of TRBP and other RNA silencing factors in cells. Our results showed the regulation of persistent BoDV infection by RNA-silencing factors in human cells. • An RNA silencing enhancer, TRBP, increased BoDV RNA levels in human cells. • TRBP interacted with BoDV RNA. • BoDV did not convert the localization of TRBP and other RNA silencing factors. [ABSTRACT FROM AUTHOR]

Self-assembled enantiomers of an asymmetric di-iron metallohelix differ in their antiproliferative activities against HCT116 colon cancer cells such that the compound with Λ-helicity at the metals becomes more potent than the Δ compound with increasing exposure time. From concentration- and temperature-dependent 57Fe isotopic labelling studies of cellular accumulation we postulate that while the more potent Λ enantiomer undergoes carrier-mediated efflux, for Δ the process is principally equilibrative. Cell fractionation studies demonstrate that both enantiomers localise in a similar fashion; compound is observed mostly within the cytoskeleton and/or genomic DNA, with significant amounts also found in the nucleus and membrane, but with negligible concentration in the cytosol. Cell cycle analyses using flow cytometry reveal that the Δ enantiomer induces mild arrest in the G1 phase, while Λ causes a very large dose-dependent increase in the G2/M population at a concentration significantly below the relevant IC50. Correspondingly, G2-M checkpoint failure as a result of Λ-metallohelix binding to DNA is shown to be feasible by linear dichroism studies, which indicate, in contrast to the Δ compound, a quite specific mode of binding, probably in the major groove. Further, spindle assembly checkpoint (SAC) failure, which could also be responsible for the observed G2/M arrest, is established as a feasible mechanism for the Λ helix via drug combination (synergy) studies and the discovery of tubulin and actin inhibition. Here, while the Λ compound stabilizes F-actin and induces a distinct change in tubulin architecture of HCT116 cells, Δ promotes depolymerization and more subtle changes in microtubule and actin networks. [ABSTRACT FROM AUTHOR]

Penaeus vannamei, experiences hypoxia in its natural habitat and in aquaculture. Under hypoxia, cells enhance anaerobic energy production through glycolysis dependent on the up-regulation of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in gills. In vertebrates, GAPDH translocates into the nucleus and displays "moonlighting" functions including apoptosis. These alternative localizations and functions have not been described in crustaceans. We examined the effect of severe hypoxia and GAPDH silencing by RNA interference (RNAi) on its mRNA expression localization and glycolytic activity in P. vannamei gills. Expression and cytosolic activity were up-regulated only in hypoxia-exposed shrimp, but not in hypoxia-silenced specimens. GAPDH was immunodetected in cytosol and nucleus regardless of oxygen conditions. Hypoxia and RNAi decreased activity in cytosol and nucleus without affecting protein abundance, which suggests that nuclear GAPDH may have non-glycolytic functions. Moreover, Caspase-3 (Casp-3) expression increased with GAPDH silencing, suggesting alternative roles for GAPDH in apoptosis evasion. [ABSTRACT FROM AUTHOR]

The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3 receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3 receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3 receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications. [ABSTRACT FROM AUTHOR]

O -GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O -GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii , and both enzymes are now recognized as O -fucosyltransferases (OFTs). Here we investigate the evolution of spy -like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium —a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma , the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation. [ABSTRACT FROM AUTHOR]

Plant viruses depend on host cellular factors for their replication and movement. There are cellular proteins that change their localization and/or expression and have a proviral role or antiviral activity and interact with or target viral proteins. Identification of those proteins and their roles during infection is crucial for understanding plant–virus interactions and to design antiviral resistance in crops. Important host proteins have been identified using approaches such as tag-dependent immunoprecipitation or yeast two hybridization that require cloning individual proteins or the entire virus. However, the number of possible interactions between host and viral proteins is immense. Therefore, an alternative method is needed for proteome-wide identification of host proteins involved in host–virus interactions. Here, we present cell fractionation coupled with mass spectrometry as an option to identify protein–protein interactions between viruses and their hosts. This approach involves separating subcellular organelles using differential and/or gradient centrifugation from virus-free and virus-infected cells (1) followed by comparative analysis of the proteomic profiles obtained for each subcellular organelle via mass spectrometry (2). After biological validation, prospect host proteins with proviral or antiviral roles can be subject to fundamental studies in the context of basic biology to shed light on both virus replication and cellular processes. They can also be targeted via gene editing to develop virus-resistant crops.

Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.

CAR-T cells are widely recognized for their potential to successfully treat hematologic cancers and provide durable response. However, severe adverse events such as cytokine release syndrome (CRS) and neurotoxicity are concerning. Our goal is to assess CAR-T cell clinical trial publications to address the question of whether administration of CAR-T cells as dose fractions reduces toxicity without adversely affecting efficacy. Systematic literature review of studies published between January 2010 and May 2022 was performed on PubMed and Embase to search clinical studies that evaluated CAR-T cells for hematologic cancers. Studies published in English were considered. Studies in children (age < 18), solid tumors, bispecific CAR-T cells, and CAR-T cell cocktails were excluded. Data was extracted from the studies that met inclusion and exclusion criteria. Review identified a total of 18 studies that used dose fractionation. Six studies used 2-day dosing schemes and 12 studies used 3-day schemes to administer CAR-T cells. Three studies had both single dose and fractionated dose cohorts. Lower incidence of Grade ≥ 3 CRS and neurotoxicity was seen in fractionated dose cohorts in 2 studies, whereas 1 study reported no difference between single and fractionated dose cohorts. Dose fractionation was mainly recommended for high tumor burden patients. Efficacy of CAR-T cells in fractionated dose was comparable to single dose regimen within the same or historical trial of the same agent in all the studies. The findings suggest that administering dose fractions of CAR-T cells over 2–3 days instead of single dose infusion may mitigate the toxicity of CAR-T cell therapy including CRS and neurotoxicity, especially in patients with high tumor burden. However, controlled studies are likely needed to confirm the benefits of dose fractionation. [ABSTRACT FROM AUTHOR]

The article highlights lustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening of 9744 lncRNAs in melanoma cells cocultured with human CD8+ T cells. It discusses that NC01198 interacts and activates NF-κB component p65 to trigger the type I and type II interferon responses in melanoma and breast cancer cells and systematically characterized novel lncRNAs involved in tumor immune response.

Background: Inactivation of the Hippo pathway promotes Yap nuclear translocation, enabling execution of a transcriptional program that induces tissue growth. Genetic lesions of Hippo intermediates only identify a minority of cancers with illegitimate YAP activation. Yap has been implicated in resistance to targeted therapies, but the mechanisms by which YAP may impact adaptive resistance to MAPK inhibitors are unknown. Methods: We screened 52 thyroid cancer cell lines for illegitimate nuclear YAP localization by immunofluorescence and fractionation of cell lysates. We engineered a doxycycline (dox)-inducible thyroid-specific mouse model expressing constitutively nuclear YAPS127A, alone or in combination with endogenous expression of either HrasG12V or BrafV600E. We also generated cell lines expressing dox-inducible sh-miR-E-YAP and/or YAPS127A. We used cell viability, invasion assays, immunofluorescence, Western blotting, qRT-PCRs, flow cytometry and cell sorting, high-throughput bulk RNA sequencing and in vivo tumorigenesis to investigate YAP dependency and response of BRAF-mutant cells to vemurafenib. Results: We found that 27/52 thyroid cancer cell lines had constitutively aberrant YAP nuclear localization when cultured at high density (NU-YAP), which rendered them dependent on YAP for viability, invasiveness and sensitivity to the YAP-TEAD complex inhibitor verteporfin, whereas cells with confluency-driven nuclear exclusion of YAP (CYT-YAP) were not. Treatment of BRAF-mutant thyroid cancer cells with RAF kinase inhibitors resulted in YAP nuclear translocation and activation of its transcriptional output. Resistance to vemurafenib in BRAF-mutant thyroid cells was driven by YAP-dependent NRG1, HER2 and HER3 activation across all isogenic human and mouse thyroid cell lines tested, which was abrogated by silencing YAP and relieved by pan-HER kinase inhibitors. YAP activation induced analogous changes in BRAF melanoma, but not colorectal cells. Conclusions: YAP activation in thyroid cancer generates a dependency on this transcription factor. YAP governs adaptive resistance to RAF kinase inhibitors and induces a gene expression program in BRAFV600E-mutant cells encompassing effectors in the NRG1 signaling pathway, which play a central role in the insensitivity to MAPK inhibitors in a lineage-dependent manner. HIPPO pathway inactivation serves as a lineage-dependent rheostat controlling the magnitude of the adaptive relief of feedback responses to MAPK inhibitors in BRAF-V600E cancers. [ABSTRACT FROM AUTHOR]

The liver is important in the synthesis, metabolism and storage of nutrients, detoxification and immune response of the body, and the liver immune response against exogenous pathogens from the intestinal tract plays a key role in the immune activities. However, the cellular composition of the liver immune atlas remains sparsely studied in reptiles. We used single-cell RNA sequencing to identify the cellular profile of the liver of the Chinese soft-shelled turtle (Pelodiscus sinensis). We obtained the transcriptional landscape based on 9938 cells from the fractionation of fresh hepatic tissues from two individuals, uninfected and infected with bacteria (Aeromonas hydrophila). We identified seven hepatic immune cell subsets, including plasma, erythroid, T/NK, B, endothelial, dendritic and Kupffer cells. Bacteria-infection altered the number of liver immune cells, as revealed by the fact that the infected turtle had more plasma, endothelial and Kupffer cells and fewer T/NK, dendritic and erythroid cells than did the uninfected turtle. Our study is the first to provide a comprehensive view of the hepatic immune landscape of P. sinensis at the single-cell resolution that outlines the characteristics of immune cells in the turtle liver and provides a liver transcriptome baseline for turtle immunology. [Display omitted] • Seven liver immune cell subsets were identified in the turtle Pelodiscus sinensis. • Bacteria-infection altered the number of liver immune cells in the turtle. • This is the first live immune landscape from turtles at the single-cell resolution. [ABSTRACT FROM AUTHOR]

Summary: Light regulates the subcellular localization of plant photoreceptors, a key step in light signaling. Ultraviolet‐B radiation (UV‐B) induces the plant photoreceptor UV RESISTANCE LOCUS 8 (UVR8) nuclear accumulation, where it regulates photomorphogenesis. However, the molecular mechanism for the UV‐B‐regulated UVR8 nuclear localization dynamics is unknown.With fluorescence recovery after photobleaching (FRAP), cell fractionation followed by immunoblotting and co‐immunoprecipitation (Co‐IP) assays we tested the function of UVR8‐interacting proteins including CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), REPRESSOR OF UV‐B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 in the regulation of UVR8 nuclear dynamics in Arabidopsis thaliana.We showed that UV‐B‐induced rapid UVR8 nuclear translocation is independent of COP1, which previously was shown to be required for UV‐B‐induced UVR8 nuclear accumulation. Instead, we provide evidence that the UV‐B‐induced UVR8 homodimer‐to‐monomer photo‐switch and the concurrent size reduction of UVR8 enables its monomer nuclear translocation, most likely via free diffusion. Nuclear COP1 interacts with UV‐B‐activated UVR8 monomer, thereby promoting UVR8 nuclear retention. Conversely, RUP1and RUP2, whose expressions are induced by UV‐B, inhibit UVR8 nuclear retention via attenuating the UVR8–COP1 interaction, allowing UVR8 to exit the nucleus.Collectively, our data suggest that UV‐B‐induced monomerization of UVR8 promotes its nuclear translocation via free diffusion. In the nucleus, COP1 binding promotes UVR8 monomer nuclear retention, which is counterbalanced by the major negative regulators RUP1 and RUP2. [ABSTRACT FROM AUTHOR]

The article discusses a 2022 development of the nitroreductase-instructed supramolecular self-assembly for the up-regulated activity of nitroreductase in F.nucleatum-infected tumors. Topics covered include these assemblies' alleviation of drug resistance and sensitizing of colorectal cancer (CRC) cells against chemo-drugs, and possible inhibition of tumor growth. Also noted is the study's broadening of the toolbox of enzyme-instructed supramolecular self-assembly.