Subcellular Fractionation Analysis Research Articles

Rhizophagus intraradices mediated mitigation of arsenic toxicity in wheat involves differential distribution of arsenic in subcellular fractions and modulated expression of Phts and ABCCs

Biomagnification of arsenic in food chain through wheat consumption poses a serious threat to human health. Therefore, it is necessary to elucidate mechanism of arsenic tolerance and detoxification in wheat. The study aimed to unravel the strategies adopted by arbuscular mycorrhizal fungi to alleviate arsenic toxicity in wheat. To accomplish this, independent and interactive effects of arsenic and Rhizophagus intraradices were assessed. Colonization by R. intraradices resulted in lower expression of high-affinity phosphate transporters (Phts) in comparison with non-mycorrhizal (NM) plants, thereby lowering arsenic concentrations in mycorrhizal (M) plants. Additionally, the subcellular fractionation analysis indicated differential distribution of arsenic. In NM plants, arsenic accumulated primarily in cell wall and organelle fractions. Conversely, in M plants arsenic was more concentrated in the cell wall and vacuolar fractions. This was related to higher levels of hydroxyl and aldehyde groups in cell wall fraction of root along with increased expression of C-type ATP-binding cassette transporters in root and leaves. These factors enabled effective sequestration of arsenic in the cell wall and vacuoles of M plants, thereby reducing its toxicity. Furthermore, the proportion of inorganic arsenic was lower in M plant, as it transformed it into less toxic organic forms.

8700 Mono(ADP-Ribosyl)ation of Ribosomal Proteins Promotes Differentiation During Adipogenesis

Abstract Disclosure: X. Tan: None. S. Challa: None. C.V. Camacho: None. T.S. Nandu: None. M.S. Stokes: None. W.L. Kraus: Other; Self; W.L.K. is a founder and stockholder for Ribon Therapeutics, Inc; a founder, member of the SAB, member of the BOD, and a stockholder for and ARase Therapeutics, Inc. He is also coholder of U.S. Patent. ADP-ribosylation, a crucial post-translational modification, involves the covalent attachment of ADP-ribose units to amino acids on proteins through ADP-ribosyl transferase (ART) enzymes, utilizing NAD+ synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs). We previously showed that NMNAT-2, a cytoplasmic NAD+ synthase, increases during adipogenesis, leading to reduced nuclear NAD+ levels through compartmentalized NMN depletion and enhanced differentiation of preadipocytes. However, the implications of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes remain unknown. Our current findings demonstrate elevated ribosome MARylation during 3T3-L1 preadipocyte differentiation, driven by NMNAT-2 induction. Inhibiting NAD+ biosynthesis by knockdown of NMNAT-2 or FK866 results in reduced ribosome MARylation, with PARP16 identified as the primary ART mediating MARylation. Knockdown of NMNAT-2 or PARP16 impaired global protein synthesis and preadipocyte differentiation. Treatment with the PARP16 inhibitor DB008 hindered ribosomal MARylation and preadipocyte differentiation. Immunofluorescence and subcellular fractionation analysis demonstrated that MARylated ribosomes are mostly associated with the endoplasmic reticulum. Using TMT mass spectrometry, we identified numerous high-confidence ADP-ribosylation sites on glutamate and aspartate residues in both undifferentiated and differentiated cells. We examined five proteins with specific sites of MARylation that are consistently more modified in the differentiated state. Among these, ADP-ribosylation of RPSA significantly promote preadipocyte differentiation; mutation of the site inhibits this effect. Ongoing investigations involve studying the impact of Parp16 knockout on high fat diet-induced obesity in mice to further determine the biological role of ribosome protein MARylation during adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and the DOD OCRP (OC200311). Presentation: 6/1/2024

Characterization of a membrane toxin-antitoxin system, tsaAT, from Staphylococcus aureus.

Bacterial toxin-antitoxin (TA) systems consist of a toxin that inhibits essential cellular processes, such as DNA replication, transcription, translation, or ATP synthesis, and an antitoxin neutralizing their cognate toxin. These systems have roles in programmed cell death, defense against phage, and the formation of persister cells. Here, we characterized the previously identified Staphylococcus aureus TA system, tsaAT, which consists of two putative membrane proteins: TsaT and TsaA. Expression of the TsaT toxin caused cell death and disrupted membrane integrity, whereas TsaA did not show any toxicity and neutralized the toxicity of TsaT. Furthermore, subcellular fractionation analysis demonstrated that both TsaA and TsaT localized to the cytoplasmic membrane of S. aureus expressing either or both 3xFLAG-tagged TsaA and 3xFLAG-tagged TsaT. Taken together, these results demonstrate that the TsaAT TA system consists of two membrane proteins, TsaA and TsaT, where TsaT disrupts membrane integrity, ultimately leading to cell death. Although sequence analyses showed that the tsaA and tsaT genes were conserved among Staphylococcus species, amino acid substitutions between TsaT orthologs highlighted the critical role of the 6th residue for its toxicity. Further amino acid substitutions indicated that the glutamic acid residue at position 63 in the TsaA antitoxin and the cluster of five lysine residues in the TsaT toxin are involved in TsaA's neutralization reaction. This study is the first to describe a bacterial TA system wherein both toxin and antitoxin are membrane proteins. These findings contribute to our understanding of S. aureus TA systems and, more generally, give new insight into highly diverse bacterial TA systems.

Exosomal LINC00958 maintains ovarian cancer cell stemness and induces M2 macrophage polarization via Hedgehog signaling pathway and GLI1 protein

Long non-coding RNA (lncRNA) LINC00958 has been reported to promote many gynecological cancers, but its detailed function in OC remains unclear. Cancer stem cells (CSCs) and tumor-associated macrophages (TAMs) have been reported to participate in the occurrence and metastasis of cancers. We want to explore the effects of exosomal LINC00958 on cell stemness and macrophage polarization in OC. LINC00958 expression was first verified in OC cells and its function on cell stemness was verified by subcellular fractionation analysis, sphere formation assay and so on. Exosomal LINC00958 was secreted from OC cells and the model of M2 macrophage polarization was established to further verify the impact of exosomal LINC00958 on the cell stemness and macrophage polarization of OC cells using several mechanism experiments including flow cytometry, RNA pulldown, luciferase reporter assays and so on. LINC00958 was up-regulated in OC cells and exosomal LINC00958 enhanced the stem cell-like properties of OC cells and M2 macrophage polarization. Furthermore, LINC00958 combined with glioma-associated oncogene homolog 1 (GLI1) to activate Hedgehog pathway, thereby promoting M2 polarization. Exosomal LINC00958 maintained OC cell stemness and induced M2 polarization via the Hedgehog signaling pathway.

CircRNA-0015004 act as a ceRNA to promote RCC2 expression in hepatocellular carcinoma

Although circular RNAs (circRNA) have been demonstrated to modulate tumor initiation and progression, their roles in the proliferation of hepatocellular carcinoma (HCC) are still poorly understood. Based on the analysis of GEO data (GSE12174), hsa-circRNA-0015004 (circ-0015004) was screened and validated in 80 sets of HCC specimens. Subcellular fractionation analysis was designed to determine the cellular location of circ-0015004. Colony formation and cell counting kit-8 were performed to investigate the role of circ-0015004 in HCC. Dual-luciferase reporter gene assays, RNA immunoprecipitation and chromatin immunoprecipitation were employed to verify the interaction among circ-0015004, miR-330-3p and regulator of chromatin condensation 2 (RCC2). The expression level of circ-0015004 was significantly upregulated in HCC cell lines and HCC tissues. HCC patients with higher circ-0015004 levels displayed shorter overall survival, and higher tumor size and TNM stage. Moreover, knockdown of circ-0015004 significantly reduced HCC cell proliferation in vitro and inhibited the growth of HCC in nude mice. Mechanistic studies revealed that circ-0015004 could upregulate the expression of RCC2 by sponging miR-330-3p, thereby promoting HCC cell proliferation. Furthermore, we identified that Ying Yang 1 (YY1) could function as an important regulator of circ-0015004 transcription. This study systematically demonstrated the novel regulatory signaling of circ-0015004/miR-330-3p/RCC2 axis in promoting HCC progression, providing insight into HCC diagnosis and treatment from bench to clinic.

Proteasome localization and activity in pig brain and in vivo small molecule screening for activators.

Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology. Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes. By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased. This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.

Abstract B015: KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth

Abstract Epithelial-to-mesenchymal transition (EMT) or its reversal mesenchymal-to-epithelial transition (MET) are of critical importance throughout development and is implicated in wound healing, fibrotic disorders, and cancer metastasis. The ability of a cell to undergo EMT and MET, cellular plasticity, underlies tumor progression through multi-step metastasis and acquisition of chemotherapy resistance as alterations in the cellular and molecular level confer changes in motility, stemness properties, gene expression, and cellular dynamics. These changes facilitate invasion and dissemination of cancer cells. Epigenetic modifications leading to cancer cell plasticity are partially driven by histone modifying proteins including lysine (K)-specific demethylase 6A (KDM6A). KDM6A is a member of the COMPASS-like protein complex, and catalyzes the removal of methyl groups from H3K27me3, facilitating gene expression. KDM6A regulation of H3K27me3 has been associated with bivalent programs in gene expression, associates with oncogenic pathways, and contributes to low survival prognoses in breast cancer. There is critical need to elucidate the mechanisms of KDM6A suppression to understand its control on cellular plasticity. Previously we have shown that KDM6A is suppressed upon EMT. In this study, we sought to identify the regulatory mechanisms of KDM6A expression and how its activity is required to maintain epithelial cellular identity. To investigate the role of KDM6A in the context of EMT, KDM6A expression was compromised using CRISPR-Cas9 to determine the impact on H3K27me3, gene expression and cellular identity. Breast cancer cell lines were transduced with CRISPR-Cas9 and GFP via lentiviral transduction. KDM6A KO and control cell lines were generated via a CRISPR KO. We observed that the KDM6A suppression or inhibition profoundly impacts cellular identity, leading to gain of mesenchymal and stemness properties. Moreover, KDM6A was determined to be regulated through sub-cellular localization leading to partial sequestration of COMPASS-like protein complex outside the nucleus. These findings were validated via Western blot analysis of subcellular protein fractions of breast cancer cell lines and KDM6A KO cell lines and well as immunofluorescence and enzyme activity analysis. We conclude that KDM6A functions as a master regulator of epithelial cellular identity which is controlled by multi-factorial mechanisms including transcriptional suppression and altered protein localization. Citation Format: Charli Worth, Provas Das, Kelsey Johnson, Joseph Taube. KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B015.

Differential protein analysis of saline-alkali promoting the oil accumulation in Nitzschia palea

BackgroundThe increasingly severe salinization of the aquatic environment has led to serious damage to the habitats of aquatic organisms. Benthic diatoms are commonly employed as indicator species for assessing water quality and serve as a reflection of the overall health of the aquatic ecosystem. Nitzschia palea is a common diatom found in freshwater, with high oil content, rapid reproductive rate, and it is a commonly dominant species in various rivers.ResultsThe results showed that after 4 days (d) of saline-alkali stress, the cell density and chlorophyll a content of Nitzschia palea reached their maximum values. Therefore, we selected Nitzschia palea under 4 d stress for Tandem Mass Tag (TMT) quantitative proteomic analysis to explore the molecular adaptation mechanism of freshwater diatoms under saline-alkali stress. Totally, 854 proteins were enriched, of which 439 differentially expressed proteins were identified. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and subcellular fractionation analysis revealed that these proteins were mainly enriched in the photosynthesis pathway, citric acid cycle (TCA cycle), fatty acid synthesis, and glutathione cycle.ConclusionsThis study aims to reveal the physiological, biochemical and proteomic mechanisms of salt and alkali tolerance and molecular adaptation of Nitzschia palea under different saline-alkali concentrations. This study showed that Nitzschia palea is one candidate of the environmental friendly, renewable bioenergy microalgae. Meantime, Nitzschiapalea reveals for the proteome of the freshwater and provides the basis, it became a model algal species for freshwater diatoms.

LINC01134 Directly Binds and Regulates SLC1A5 Stability to Promotes Colorectal Cancer Progression.

Background: Colorectal cancer (CRC) is a common malignant tumor with a poor prognosis. Long noncoding RNAs (lncRNAs) have recently gained attention for their pivotal role in regulating cancer progression, including CRC. This study aimed to investigate the biological mechanisms underlying the participation of long intergenic non-protein coding RNA 1134 (LINC01134) in the progression of CRC. Material and Methods: Quantitative Real-time-PCR (RT-qPCR) and western blot were applied to assess the expression levels of mRNA and protein. Functional experiments (CCK8 assay, colon formation assay, EdU assay and flow cytometry) were applied to assess cell viability and apoptosis. RNA-RNA interaction assays, subcellular fractionation analysis and dual luciferase reporter assays were employed to explore molecular interactions between LINC01134 and solute carrier family 1 member 5 (SLC1A5). The mRNA stability was analyzed using actinomycin D (ActD). Results: We found that LINC01134 expression was highly expressed in CRC tissues and positively correlated with advanced clinical stages and unfavorable prognosis, which is consistent with findings from CRC cell lines. Functional experiments showed that suppressing LINC01134 restrained the proliferation of CRC both in vitro and in vivo and induced apoptosis of CRC cells. Gene co-expression analysis revealed a positive relationship between LINC01134 and SLC1A5, which was also upregulated and associated with unfavorable prognosis in CRC. Further analysis of RNA interactions and mRNA stability revealed that LINC01134 directly binds to SLC1A5 mRNA, enhancing its stability. Remarkably, silencing SLC1A5 expression partially counteracted the promotion of CRC cell proliferation by LINC01134 overexpression and alleviated its inhibition of apoptosis. Conclusions: Our findings indicated that LINC01134 functioned as an oncogene in CRC by binding directly to SLC1A5 mRNA and increasing its stability. Therefore, targeting LINC01134 could be a potential therapeutic target for treating CRC.

Glutathione transferase P1 is modified by palmitate.

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Comparative proteomic analysis of vancomycin-sensitive and vancomycin-intermediate resistant Staphylococcus aureus.

The extensive use of vancomycin has led to the development of Staphylococcus aureus strains with varying degrees of resistance to vancomycin. The present study aimed to explore the molecular causes of vancomycin resistance by conducting a proteomics analysis of subcellular fractions isolated from vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-sensitive S. aureus (VSSA) strains. We conducted proteomics analysis of subcellular fractions isolated from 2 isogenic S. aureus strains: strain 11 (VSSA) and strain 11Y (VISA). We used an integrated quantitative proteomics approach assisted by bioinformatics analysis, and comprehensively investigated the proteome profile. Intensive bioinformatics analysis, including protein annotation, functional classification, functional enrichment, and functional enrichment-based cluster analysis, was used to annotate quantifiable targets. We identified 128 upregulated proteins and 21 downregulated proteins in strain 11Y as compared to strain 11. The largest group of differentially expressed proteins was composed of enzymatic proteins associated with metabolic and catalytic activity, which accounted for 32.1% and 50% of the total proteins, respectively. Some proteins were indispensable parts of the regulatory networks of S. aureus that were altered with vancomycin treatment, and these proteins were related to cell wall metabolism, cell adhesion, proteolysis, and pressure response. Our proteomics study revealed regulatory proteins associated with vancomycin resistance in S. aureus. Some of these proteins were involved in the regulation of cell metabolism and function, which provides potential targets for the development of strategies to manage vancomycin resistance in S. aureus.

LncRNA ARAP1-AS1 enhances proliferation and impairs apoptosis of lymphoma cells by sponging miR-6867-5p.

Numerous evidence have suggested the vital role of lncRNAs in human tumorigenesis. And lncRNA APAP1-AS1 has been proved to act as an oncogene. Nevertheless, the molecular process underlying ARAP1-AS1 for the lymphoma progression has not been well studied. RT-qPCR was used to ascertain the miR-6867-5p and ARAP1-AS1 in lymphoma cells and tissues. The localization of ARAP1-AS1 was determined via subcellular fractionation analysis. A xenograft model was used to investigate the influence of ARAP1-AS1 in formation of tumor in vivo. In addition, interactions between ARAP-AS1 and miR-6867-5p were tested by bioinformatics analysis, RIP assay, luciferase reporter and Pearson's correlation analysis. Combined with loss-of-function experiments, MTT assays and flow cytometry were performed to evaluate the function of miR-6867-5p and also ARAP-AS1 in proliferation and apoptosis of lymphoma cells, respectively. ARAP1-AS1 was remarkably upregulated in lymphoma cells and tissues, while miR-6867-5p expression was downregulated. Furthermore, high ARAP1-AS1 expression suppressed miR-6867-5p expression in lymphoma cell lines (Raji and CA46), and Pearson's analysis showed negative correlation between ARAP1-AS1 expression and also miR-6867-5p expression. In addition, knockdown of ARAP1-AS1 resulted in weakened cell viability and uplifted apoptosis rate of lymphoma cells (Raji and CA46) as well as a delay in the tumor growth in vivo. Further investigations illustrated that miR-6867-5p inhibitor reversed all above biological activities. LncRNA ARAP1-AS1 served as a tumor-promoter in lymphoma cells by sponging with miR-6867-5p, which may help to provide potential therapeutic target gene for lymphoma patients.

THU544 Prostatic Acid Phosphatase Is Negatively Regulated By Androgens And Persists In Castrate-resistant Metastatic Prostate Cancer

Abstract Disclosure: V. Rajagopalan: None. A. Kirschenbaum: None. S. Yao: None. A. Levine: None. Abstract: Prostate cancer (PCa) is the most common cancer and second leading cause of cancer death in American men. Most patients with metastatic PCa respond initially to androgen deprivation therapy (ADT) but the majority will progress to lethal, metastatic, castrate-resistant PCa (mCRPC). The identification of markers of mCRPC that are not regulated or negatively regulated by androgens can serve as markers and therapeutic targets in mCRPC. We hypothesized that prostatic acid phosphatase (PAP), a protein phosphatase and 5’ectonucleotidase that is expressed in both the cytoplasm and transmembrane (TM-PAP) of PCa cells, is negatively regulated by androgen signaling and can serve as a therapeutic target. Methods: Protein expression of PAP was assessed with western blotting in PCa cell lines. TM-PAP is identified by sub-cellular fractionation analysis, confocal microscopy, and flow cytometry. Effects of androgens and anti-androgens on PAP expression were assessed in VCaP cells with dihydrotestosterone (DHT) and Enzalutamide treatment followed by PAP, AR and PSA protein expression determined with Western Blotting. Loss of function analysis of PAP was performed with siRNA-mediated silencing and functional analysis was evaluated with migration and colony formation assays. Transcriptional regulation of PAP was determined by real time qPCR analysis. ChiPseq was done to confirm the binding of AR to PAP promoter. Results: All forms of PAP including TM-PAP are expressed in VCaP cell line. However, we did not detect PAP protein expression in-vitro in the LNCaP, C42B or 22Rv1 cell lines. In VCaP cell lines, we demonstrated that androgens decreased while anti-androgens increased PAP expression in a time and dose dependent fashion. Androgenic regulation of PAP occurred, at least in part, at the transcriptional level. We successfully knocked down TM and secretory PAP expression in VCaP cells using siRNA technology and showed that loss of PAP decreased migration and colony formation. Conclusions: The identification of markers of mCRPC that not regulated or negatively regulated by androgen could help identify recalcitrant tumor cells and mount targeted delivery of therapeutic agents. In the VCaP cell line, derived from a patient with mCRPC, TM and cytosolic PAP is one such protein that is negatively regulated by androgen signaling and may therefore serve as a viable therapeutic target in mCRPC. Presentation: Thursday, June 15, 2023

Analysis of subcellular RNA fractions demonstrates significant genetic regulation of gene expression in human brain post-transcriptionally

Gaining insight into the genetic regulation of gene expression in human brain is key to the interpretation of genome-wide association studies for major neurological and neuropsychiatric diseases. Expression quantitative trait loci (eQTL) analyses have largely been used to achieve this, providing valuable insights into the genetic regulation of steady-state RNA in human brain, but not distinguishing between molecular processes regulating transcription and stability. RNA quantification within cellular fractions can disentangle these processes in cell types and tissues which are challenging to model in vitro. We investigated the underlying molecular processes driving the genetic regulation of gene expression specific to a cellular fraction using allele-specific expression (ASE). Applying ASE analysis to genomic and transcriptomic data from paired nuclear and cytoplasmic fractions of anterior prefrontal cortex, cerebellar cortex and putamen tissues from 4 post-mortem neuropathologically-confirmed control human brains, we demonstrate that a significant proportion of genetic regulation of gene expression occurs post-transcriptionally in the cytoplasm, with genes undergoing this form of regulation more likely to be synaptic. These findings have implications for understanding the structure of gene expression regulation in human brain, and importantly the interpretation of rapidly growing single-nucleus brain RNA-sequencing and eQTL datasets, where cytoplasm-specific regulatory events could be missed.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling

BackgroundHeat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. ObjectivesIn this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. MethodsHSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. ResultsUsing subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2β1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. ConclusionThe present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

High-Coverage Strategy for Multi-Subcellular Metabolome Analysis Using Dansyl-Labeling-Based LC-MS/MS.

Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge. Herein, we present a comprehensive subcellular fractionation, characterization, and metabolome analysis strategy. First, six subcellular fractions including nuclei, mitochondria, lysosomes, peroxisomes, microsomes, and cytoplasm were generated from a single aliquot of liver homogenate. Then, a dansyl-labeling-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 151 amino/phenol- or carboxyl-containing metabolites in the subcellular fractions was established and validated. Last, the strategy was applied to a rat model of carbon tetrachloride (CCl4)-induced acute liver injury (ALI). The metabolic profile of individual organelles was compared with that of the liver. Interestingly, many unique changes were observed specifically in organelles, while the liver failed to capture these changes. This result indicates that metabolic investigation at the tissue level might lead to erroneous results due to the leveling effect. Our study demonstrates a feasible approach for the broad-spectrum-targeted metabolic profiling of multi-subcellular fractions, which can be of great use in driving our further understanding of intracellular metabolic activities in various physical and pathological conditions.

Cadmium tolerance in Elodea canadensis Michx: Subcellular distribution and metabolomic analysis

The aquatic plant Elodea canadensis is considered a good candidate for ecotoxicological investigations. Cadmium (Cd) is a widespread contaminant in aquatic systems. In this study, to better elucidate the underlying tolerance mechanism and molecular impact of environmentally relevant Cd concentration in aquatic plants, subcellular distribution, chemical forms, and gas chromatography–mass spectrometry-based non-targeted metabolomics profiles were comprehensively analyzed in E. canadensis subjected to 0 and 10 µM Cd treatment for 5 d. Subcellular fractionation analysis of Cd-containing leaves showed that 67% of Cd was compartmentalized in cell wall followed by the soluble fraction (24 %) and organelles (9 %). The majority of Cd (90 %) was found in the extraction using 1 M NaCl. Metabolomic analysis using unsupervised principal component analyses and a supervised partial least squares discriminant analysis revealed clear differences in metabolic profiles between the two groups, demonstrating the metabolic effects of Cd. The 155 identified compounds altered by Cd were mainly from primary metabolism, including sugars, amino acids, organic acids, and their derivatives. Secondary metabolites such as polyphenols and phenolamides were also detected. The massive up-regulation of metabolites, including trehalose, proline, sarcosine, nicotianamine, putrescine, α-ketoglutaric acid, citric acid, and phytol might represent a detoxification mechanism. These findings highlighted the mechanistic strategies that E. canadensis employs to defend against Cd toxicity.

Early Alterations of RNA Binding Protein (RBP) Homeostasis and ER Stress-Mediated Autophagy Contributes to Progressive Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa (RP).

The retinal degeneration 10 (rd10) mouse model is widely used to study retinitis pigmentosa (RP) pathomechanisms. It offers a rather unique opportunity to study trans-neuronal degeneration because the cell populations in question are separated anatomically and the mutated Pde6b gene is selectively expressed in rod photoreceptors. We hypothesized that RNA binding protein (RBP) aggregation and abnormal autophagy might serve as early pathogenic events, damaging non-photoreceptor retinal cell types that are not primarily targeted by the Pde6b gene defect. We used a combination of immunohistochemistry (DAB, immunofluorescence), electron microscopy (EM), subcellular fractionation, and Western blot analysis on the retinal preparations obtained from both rd10 and wild-type mice. We found early, robust increases in levels of the protective endoplasmic reticulum (ER) calcium (Ca2+) buffering chaperone Sigma receptor 1 (SigR1) together with other ER-Ca2+ buffering proteins in both photoreceptors and non-photoreceptor neuronal cells before any noticeable photoreceptor degeneration. In line with this, we found markedly altered expression of the autophagy proteins p62 and LC3, together with abnormal ER widening and large autophagic vacuoles as detected by EM. Interestingly, these changes were accompanied by early, prominent cytoplasmic and nuclear aggregation of the key RBPs including pTDP-43 and FET family RBPs and stress granule formation. We conclude that progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy, along with abnormal cytoplasmic RBP aggregation.

The Long Noncoding RNA LINC00958 Is Induced in Psoriasis Epidermis and Modulates Epidermal Proliferation

Psoriasis is a common, immune-mediated skin disease characterized by epidermal hyperproliferation and chronic skin inflammation. Long noncoding RNAs are >200 nucleotide-long transcripts that possess important regulatory functions. To date, little is known about the contribution of long noncoding RNAs to psoriasis. In this study, we identify LINC00958 as a long noncoding RNA overexpressed in keratinocytes (KCs) from psoriasis skin lesions, in a transcriptomic screen performed on KCs sorted from psoriasis and healthy skin. Increased levels of LINC00958 in psoriasis KCs were confirmed by RT-qPCR and single-molecule in situ hybridization. Confocal microscopy and analysis of subcellular fractions showed that LINC00958 is mainly localized in the cytoplasm of KCs. IL-17A, a key psoriasis cytokine, induced LINC00958 in KCs through C/EBP-β and the p38 pathway. The inhibition of LINC00958 led to decreased proliferation as measured by Ki-67 expression, live cell analysis imaging, and 5-ethynyl-2-deoxyuridine assays. Transcriptomic analysis of LINC00958-depleted KCs revealed enrichment of proliferation- and cell cycle‒related genes among differentially expressed transcripts. Moreover, LINC00958 depletion led to decreased basal and IL-17A‒induced phosphorylation of p38. Furthermore, IL-17A‒induced KC proliferation was counteracted by the inhibition of LINC00958. In summary, our data support a role for the IL-17A‒induced long noncoding RNA, LINC00958, in the pathological circuits of psoriasis by reinforcing IL-17A‒induced epidermal hyperproliferation.

Tumor-secreted exosomal miR-141 activates tumor-stroma interactions and controls premetastatic niche formation in ovarian cancer metastasis

BackgroundMetastatic colonization is one of the critical steps in tumor metastasis. A pre-metastatic niche is required for metastatic colonization and is determined by tumor-stroma interactions, yet the mechanistic underpinnings remain incompletely understood.MethodsPCR-based miRNome profiling, qPCR, immunofluorescent analyses evaluated the expression of exosomal miR-141 and cell-to-cell communication. LC-MS/MS proteomic profiling and Dual-Luciferase analyses identified YAP1 as the direct target of miR-141. Human cytokine profiling, ChIP, luciferase reporter assays, and subcellular fractionation analyses confirmed YAP1 in modulating GROα production. A series of in vitro tumorigenic assays, an ex vivo model and Yap1 stromal conditional knockout (cKO) mouse model demonstrated the roles of miR-141/YAP1/GROα/CXCR1/2 signaling cascade. RNAi, CRISPR/Cas9 and CRISPRi systems were used for gene silencing. Blood sera, OvCa tumor tissue samples, and tissue array were included for clinical correlations.ResultsHsa-miR-141-3p (miR-141), an exosomal miRNA, is highly secreted by ovarian cancer cells and reprograms stromal fibroblasts into proinflammatory cancer-associated fibroblasts (CAFs), facilitating metastatic colonization. A mechanistic study showed that miR-141 targeted YAP1, a critical effector of the Hippo pathway, reducing the nuclear YAP1/TAZ ratio and enhancing GROα production from stromal fibroblasts. Stromal-specific knockout (cKO) of Yap1 in murine models shaped the GROα-enriched microenvironment, facilitating in vivo tumor colonization, but this effect was reversed after Cxcr1/2 depletion in OvCa cells. The YAP1/GROα correlation was demonstrated in clinical samples, highlighting the clinical relevance of this research and providing a potential therapeutic intervention for impeding premetastatic niche formation and metastatic progression of ovarian cancers.ConclusionsThis study uncovers miR-141 as an OvCa-derived exosomal microRNA mediating the tumor-stroma interactions and the formation of tumor-promoting stromal niche through activating YAP1/GROα/CXCRs signaling cascade, providing new insight into therapy for OvCa patients with peritoneal metastases.