Intracellular Protein Transport Research Articles

Evaluating Cell Membrane Localization and Intracellular Transport of Proteins by Biotinylation.

Protein translocation to the cell membrane and transport through intracellular compartments are dynamic processes frequently altered in cancer cells. Abnormal protein localization can affect key cell functions, including transduction of extracellular signals and organization of the cytoskeleton, significantly affecting oncogenicity and therapeutic responses. In this chapter, we describe a surface protein biotinylation method that allows the study of membrane localization and endosomal transport of membrane-associated proteins. Surface biotinylation can be used to evaluate baseline protein levels at the membrane, and other processes such as internalization, recycling, and degradation of proteins in response to different treatments or as a consequence of oncogenic mutations. Further, the combination of this technique with other strategies, such as treatments with transport inhibitors, allows investigation of specific steps of protein trafficking through the cell.

Cyclic fertilin-derived peptide stimulates in vitro human embryo development

To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin β (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor. Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts. The study was performed in an academic hospital and research laboratory. Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine." Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action. Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 μM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the invitro hatching rate from 14% to 45%. Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport. Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the invitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.

Involvement of Rab25 Biomarker in Pathogenesis of Ovarian Cancer

Ovarian tumor is the third leading common gynecologic tumor and the common leading cause of death in gynecological cancers to the entire global and studies suggested that Rab25 is insinuated in the pathological process of ovarian cancer. Despite the availability of biomarkers for ovarian cancer detection, there are no specific markers that enable the early detection of ovarian cancers which open an avenue to Rab25 to be review. A number of genes and proteins have been reported to be involved in the pathogenesis of ovarian cancers. Of them, Ras-related protein 25 (Rab25) is suggested to be linked to increased risk of ovarian cancer development. Rab25, an intracellular transport protein, belongs to the Rab small GTPase family and regulates various aspects of internalized membrane protein recycling and trafficking occurring inside the cells to the cell membrane. It is known to be involved in cell proliferation, and prevents apoptosis and invasion in ovarian cancer. Rab25 is highly found in epithelial cells and the expression of Rab25 proteins has been implicated to be ubiquitous. Upregulation of Rab25 has also been strongly shown to intensify the cancer cell proliferation and to prevent apoptosis in vitro and in vivo .Here in we will review the past and current studies implicating Rab 25 as potential biomarker in ovarian cancer in addition to pathogenesis

The Role of GM130 in Nervous System Diseases.

Golgi matrix protein 130 (GM130) is a Golgi-shaping protein located on the cis surface of the Golgi apparatus (GA). It is one of the most studied Golgin proteins so far. Its biological functions are involved in many aspects of life processes, including mitosis, autophagy, apoptosis, cell polarity, and directed migration at the cellular level, as well as intracellular lipid and protein transport, microtubule formation and assembly, lysosome function maintenance, and glycosylation modification. Mutation inactivation or loss of expression of GM130 has been detected in patients with different diseases. GM130 plays an important role in the development of the nervous system, but the studies on it are limited. This article reviewed the current research progress of GM130 in nervous system diseases. It summarized the physiological functions of GM130 in the occurrence and development of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), microcephaly (MCPH), sepsis associated encephalopathy (SAE), and Ataxia, aiming to provide ideas for the further study of GM130 in nervous system disease detection and treatment.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses.

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

ABSTRACTThe intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21–183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21–183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions.

P–294 Mapping COVID–19 affected genes from blood in a Window of implantation co-expression network reveals a potentially compromised landscape

Study questionCould the transcriptomic and functional landscape of the window of implantation be compromised by SARS-COV–2 infection?Summary answerSome of the main genes and pathways involved in the window of implantation are affected in blood of COVID–19 patients and receptivity could be affected.What is known alreadyThere is a concern whether SARS-COV–2 can disrupt assisted reproduction treatments (ARTs) and fertility in short and long terms. In the endometrium, it was found that genes related to the viral infection (ACE2, TMPRSS2/4, CTSL/B) are involved in menstrual cycle progression, especially in the Window of Implantation (WOI). However, there are no studies describing the transcriptome changes after the infection, and the changes that could affect receptivity and embryo implantation. Currently transcriptomic datasets are publicly available regarding virus infection effects in blood. The aim of this study was to integrate these blood effects with the gene expression during the WOI.Study design, size, durationA public dataset with blood transcriptome of 231 female COVID–19 patients and 30 female controls was downloaded from GEO. Meanwhile, 5 transcriptomic endometrial datasets in the WOI with patients without endometrial pathologies were also retrieved (n = 44). Gene expression correlations (potential activations and inhibitions) were calculated in endometrium and filtered by blood differentially expressed genes for predicting the potential effects of COVID–19 in endometrial factor. Additionally, we discovered new endometrial genes involved in the infection repercussions.Participants/materials, setting, methodsA gene co-expression network was built in Cytoscape with the WOI dataset [Pearson correlation = 0.65, only significant correlations; Power fit law R2 > = 0.8]. Differential expression was done for COVID–19 patients versus controls with limma and significant genes in blood were highlighted in the endometrial WOI network. Topological parameters were calculated by CytoHubba and network modules and related functions were analysed performing a Functional enrichment (BINGO). Statistical significance cut off was stablished in FDR<0.05.Main results and the role of chanceAfter filtering by blood affected genes, 2051 genes were found differentially expressed in COVID–19 females in blood and mapped in the co-expression WOI network. Nine modules were highlighted being enriched in translational elongation, intracellular protein transport, endosome organization, vitamin D receptor binding, actin cytoskeleton organization, RNA splicing, among others. Important hubs in the endometrium that correlated with TMPRSS4 were: COBL, a gene that promotes formation of cell ruffles which are important or embryo adhesion (FC = –3.99, degree = 209); PKP2 (FC = –1.5, degree = 188) which could play a role in junctional plaques and knockdown in mice was reported to inhibit implantation; SOCS3, linked to unexplained infertility and pregnancy loss, (FC –4.3, degree = 177); GPX3 involved in detoxification and usually highly upregulated during WOI was downregulated (FC –3.7, degree = 173). GPX3 also correlated with CTSB. TPRC/CD45, related to unexplained pregnancy loss and concentration of NK cells, was an upregulated gene (FC = 5, degree = 161) that correlated with CTSL. Upregulated genes with main connections in the network were: SERPING1 (FC = 5), which regulates complement activation and embryo-maternal immune modulation and SMARCAD1 (FC 1.5), involved in DNA repair and heterochromatin organization.Limitations, reasons for cautionThis is an in-silico descriptive study where differentially expressed genes in blood samples of COVID–19 patients were analysed in an endometrial co-expression network context. Studying a COVID–19 infected endometrium during WOI would help to confirm the results of this study.Wider implications of the findings: Although ACE2 has been reported as not highly expressed during the WOI, this study describes potential genes and functions very important for embryo implantation affected after SARS-COV–2 infection. These findings evidenced how SARS-COV–2 could impact the efficacy of ARTs and should be taken into consideration for further research and implications.Trial registration numberNot applicable

Profile of the RNA in exosomes from astrocytes and microglia using deep sequencing: implications for neurodegeneration mechanisms.

Glial cells play an important role in signal transduction, energy metabolism, extracellular ion homeostasis and neuroprotection of the central nervous system. However, few studies have explained the potential effects of exosomes from glial cells on central nervous system health and disease. In this study, the genes expressed in exosomes from astrocytes and microglia were identified by deep RNA sequencing. Kyoto Encyclopedia of Genes and Genomes analysis indicated that several pathways in these exosomes are responsible for promoting neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Gene ontology analysis showed that extracellular exosome, mitochondrion and growth factor activity were enriched in exosomes from the unique astrocyte group, while extracellular exosome and mitochondrion were enriched in exosomes from the unique microglia group. Next, combined with the screening of hub genes, the protein-protein interaction network analysis showed that exosomes from astrocytes influence neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas exosomes from microglia influence neurodegenerative diseases through immune inflammation and oxidative stress. Although there were differences in RNA expression between exosomes from astrocytes and microglia, the groups were related by the hub genes, ubiquitin B and heat shock protein family A (Hsp70) member 8. Ubiquitin B appeared to be involved in pleiotropic regulatory functions, including immune regulation, inflammation inhibition, protein catabolism, intracellular protein transport, exosomes and oxidative stress. The results revealed the clinical significance of exosomes from glia in neurodegenerative diseases. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. S20180102-152) on January 2, 2018.

Recent ultra-rare inherited variants implicate new autism candidate risk genes.

Autism is a highly heritable complex disorder in which de novo mutation (DNM) variation contributes significantly to risk. Using whole-genome sequencing data from 3,474 families, we investigate another source of large-effect risk variation, ultra-rare variants. We report and replicate a transmission disequilibrium of private, likely gene-disruptive (LGD) variants in probands but find that 95% of this burden resides outside of known DNM-enriched genes. This variant class more strongly affects multiplex family probands and supports a multi-hit model for autism. Candidate genes with private LGD variants preferentially transmitted to probands converge on the E3 ubiquitin-protein ligase complex, intracellular transport and Erb signaling protein networks. We estimate that these variants are approximately 2.5 generations old and significantly younger than other variants of similar type and frequency in siblings. Overall, private LGD variants are under strong purifying selection and appear to act on a distinct set of genes not yet associated with autism.

A generative network model of neurodevelopmental diversity in structural brain organization

The formation of large-scale brain networks, and their continual refinement, represent crucial developmental processes that can drive individual differences in cognition and which are associated with multiple neurodevelopmental conditions. But how does this organization arise, and what mechanisms drive diversity in organization? We use generative network modeling to provide a computational framework for understanding neurodevelopmental diversity. Within this framework macroscopic brain organization, complete with spatial embedding of its organization, is an emergent property of a generative wiring equation that optimizes its connectivity by renegotiating its biological costs and topological values continuously over time. The rules that govern these iterative wiring properties are controlled by a set of tightly framed parameters, with subtle differences in these parameters steering network growth towards different neurodiverse outcomes. Regional expression of genes associated with the simulations converge on biological processes and cellular components predominantly involved in synaptic signaling, neuronal projection, catabolic intracellular processes and protein transport. Together, this provides a unifying computational framework for conceptualizing the mechanisms and diversity in neurodevelopment, capable of integrating different levels of analysis—from genes to cognition.

The relationship between adrenocortical candidate gene expression and clinical response to hydrocortisone in patients with septic shock.

To determine if adrenocortical gene expression is associated with clinical outcomes or response to corticosteroid treatment in septic shock. A pre-specified nested cohort study of a randomised controlled trial of hydrocortisone compared to placebo in septic shock. Blood was collected for RNAseq analysis prior to treatment with hydrocortisone or placebo. The expression of adrenocortical candidate genes related to pituitary releasing hormones, mineralocorticoid and glucocorticoid receptors, intracellular glucocorticoid metabolism and transport proteins was measured. From May 2014 to April 2017, 671 patients were enrolled in the nested cohort study, from which 494 samples were available for analysis. We found no evidence of an association between candidate gene expression levels and either 90-day mortality, 28-day mortality or time to shock reversal. We observed evidence of a significant interaction between expression and treatment group for time to shock reversal in two genes; GLCCI1 (HR 3.81, 95%CI 0.57-25.47 vs. HR 0.64, 95%CI 0.13-3.07 for hydrocortisone and placebo respectively, p for interaction 0.008) and BHSD1 (HR 0.55, 95%CI 0.28-1.09 vs. HR 1.32 95%CI 0.67-2.60, p for interaction 0.01). In patients with septic shock, there is no association between adrenocortical candidate gene expression and mortality. Patients with higher expression of GLCCI1 who received hydrocortisone achieved shock resolution faster than those receiving placebo; conversely, patients who had higher expression ofBHSD1who received hydrocortisone achieved shock resolutionslowerthan those who received placebo. Variation in gene expression may be a mechanism for heterogeneity of treatment response to corticosteroids in septic shock.

Nelfinavir Blocks Export of Newly Synthesized Protein from the ER and Interacts with ER-Resident and Mitochondrial Proteins in an Activity-Dependent Fashion

The majority of multiple myeloma (MM) patients initially respond to proteasome inhibitor (PI)-based therapy, but later develop PI resistance and ultimately dies from PI-refractory MM. PI sensitivity of MM cells correlates with the activation status of the unfolded protein response (UPR) and its major activating axis IRE1/XBP1. PI-refractory MM cells downregulate IRE1/XBP1, and high IRE1/XBP1 expression correlates with clinical sensitivity to PI treatment. The anti-HIV drug nelfinavir induces IRE1/XBP1 and overcomes PI-resistance of MM in vitro . Nelfinavir showed unprecedented clinical activity in PI-refractory, heavily pretreated MM with ORR 65% in combination with bortezomib and dexamethasone. The molecular target of nelfinavir and its mechanism of action in MM cells are unknown. We synthesized structurally modified nelfinavir analogues equipped with a covalently protein-binding, photo-reactive moiety and functional groups that allow affinity purification of the nelfinavir analogue bound to its interacting cellular protein (SC-441), together with appropriate controls where the active center of nelfinavir was sterically occupied (SC-451). Isolation and identification of nelfinavir-interacting proteins in AMO-1 MM cells and cells adapted to carfilzomib (AMO-CFZ) was achieved with these tools by photoactivation in living cells, affinity purification and tandem mass spectrometry. We identified a small set of proteins that specifically bound to active nelfinavir. The majority of these were endoplasmic reticulum (ER)-resident proteins involved in protein quality control and transport from ER to Golgi apparatus. In addition, individual proteins from mitochondria or the nucleus were identified. We thus hypothesized that nelfinavir interferes with intracellular protein transport, in line with its UPR-activating properties. Using a construct of ST-SBP-TNFa-EGFP engineered with an ER-retention signal (KDEL) that can be released upon biotin treatment of cells (RUSH-system), in conjunction with confocal microscopy and flow cytometry in U2OS cells, we show that nelfinavir, but not its analogue SC-451, which lacks proteasome inhibitor-sensitizing activity, retains TNFa-EGFP in the ER and prevents its transport to the Golgi and into the extracellular space. We confirmed in MM cells that active nelfinavir, but not SC-451, likewise retains newly synthesized IgA by preventing its secretion, and similarly decreases MHC class I export to the cell surface.Functionally depletion of the putative ER-targets of nelfinavir by siRNA in U2OS cells sensitized cells to nelfinavir and proteasome inhibitors. The mitochondrial targets of nelfinavir were identified as constituents of the mitochondria permeability transition pore (mPTP), a multimeric protein complex that regulates the permeability of mitochondrial membranes. The formation of reactive oxygen species (ROS) and inhibition of ABCB1 efflux, known for nelfinavir, can be functionally linked to mPTP modulation.In conclusion, we demonstrate that nelfinavir blocks export of newly synthetized protein from the ER, consistent with its activity-dependent binding to ER proteins. The block in protein export from the ER by nelfinavir is consistent with its ER stress-inducing, IRE1/XBP1-activating, and PI-sensitizing effect in the clinic. We further identify mPTP as an additional protein target of nelfinavir, consistent with its ROS-inducing and ABCB1-modulating activity. The block in ER protein export represents a novel mechanism of action to sensitize MM cells for proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.

Slow viral propagation during initial phase of infection leads to viral persistence in mice

Immune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

A nontoxigenic form of Shiga toxin 2 suppresses the production of amyloid β by altering the intracellular transport of amyloid precursor protein through its receptor-binding B-subunit

Accumulation of amyloid-β peptide (Aβ) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer’s disease (AD); therefore, inhibition of Aβ accumulation offers a promising approach for therapeutic strategies against AD. Aβ is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aβ is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aβ. mStx2a-treatment also inhibited the extracellular release of Aβ. Therefore, mStx2a may provide a new strategy to inhibit the production of Aβ by modulating the intracellular transport of APP.

Over-Expression of Intestinal Alkaline Phosphatase Attenuates Atherosclerosis.

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STING-Mediated Lung Inflammation and Beyond.

Mendelian autoinflammatory diseases characterized by constitutive activation of the type I interferon pathway, the so-called type I interferonopathies, constitute a rapidly expanding group of inborn errors of immunity. Among the type I interferonopathies, STING-associated vasculopathy with onset in infancy (SAVI) and COPA syndrome were described in the last 6years, both manifesting a major inflammatory lung component associated with significant morbidity and increased mortality. There is striking clinical and histopathological overlap between SAVI and COPA syndrome, although distinct features are also present. Of note, there is a remarkably high frequency of clinical non-penetrance among individuals harboring pathogenic COPA mutations. SAVI is caused by, principally heterozygous, gain-of-function mutations in STING1 (previously referred to as TMEM173) encoding STING, a key adaptor of the interferon signaling pathway induced by DNA. COPA syndrome results from heterozygous dominant-negative mutations in the coatomer protein subunit alpha, forming part of a complex involved in intracellular cargo protein transport between the Golgi and the endoplasmic reticulum (ER). Of importance, a role for COPA in regulating the trafficking of STING, an ER-resident protein which translocates to the Golgi during the process of its activation, was recently defined, thereby possibly explaining some aspects of the phenotypic overlap between SAVI and COPA syndrome. Here, we review the expanding phenotype of these diseases, highlighting common as well as specific features, and recent advances in our understanding of STING biology that have informed therapeutic decision-making in both conditions. Beyond these rare Mendelian disorders, DNA sensing through STING is likely relevant to the pathology of several diseases associated with lung inflammation, including systemic lupus erythematosus, dermatomyositis, environmental toxin exposure, and viral infection.

The c-terminal region of BLT2 restricts its localization to the lateral membrane in a LIN7C-dependent manner.

Leukotriene B4 receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) mainly expressed in epithelial cells, where it enhances barrier function. A unique characteristic of BLT2 is its restricted localization to the lateral membrane. However, the molecular mechanism underlying the localization of BLT2 to the lateral membrane and the physiological roles of laterally localized BLT2 are unknown. BLT1 is the most homologous GPCR to BLT2 and localizes to both the apical and lateral membranes. In this study, we generated chimeric receptors of BLT2 and BLT1 as well as deletion mutants of BLT2 to determine the region(s) of BLT2 responsible for its localization. Chimeric receptors containing the C-terminal domain of BLT2 localized only to the lateral membrane, and the C-terminal deletion mutant of BLT2 accumulated at the Golgi apparatus. Furthermore, the middle and C-terminal regions of BLT2 were important for maintaining epithelial barrier function. Proteomics analysis using the chimeric BLT-ascorbate peroxidase 2 biotinylation method showed that some proteins involved in intracellular protein transport, cell-cell junctions, and actin filament binding were located very close to the C-terminal domain of BLT2. Knockdown of lin-7 homolog C (LIN7C), a membrane trafficking protein, led to accumulation of BLT2 in the Golgi apparatus, resulting in diminished epithelial barrier function. These results suggest that the C-terminal region of BLT2 plays an important role in the transport of BLT2 from the Golgi apparatus to the plasma membrane in a LIN7C-dependent manner.

Bioinformatics analysis of miRNA and mRNA expression profiles to reveal the key miRNAs and genes in osteoarthritis

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease and the most frequent type of arthritis. This study aimed to identify the key miRNAs and genes associated with OA progression.MethodsThe GSE105027 (microRNA [miRNA/miR] expression profile; 12 OA samples and 12 normal samples) and GSE48556 (messenger RNA [mRNA] expression profile; 106 OA samples and 33 normal samples) datasets were selected from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and miRNAs (DEMs) were analyzed using the limma and ROCR packages in R, respectively. The target genes that negatively correlated with the DEMs were predicted, followed by functional enrichment analysis and construction of the miRNA-gene and miRNA-transcription factor (TF)-gene regulatory networks. Additionally, key miRNAs and genes were screened, and their expression levels were verified by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsA total of 1696 DEGs (739 upregulated and 957 downregulated) and 108 DEMs (56 upregulated and 52 downregulated) were identified in the OA samples. Furthermore, 56 target genes that negatively correlated with the DEMs were predicted and found to be enriched in three functional terms (e.g., positive regulation of intracellular protein transport) and three pathways (e.g., human cytomegalovirus infection). In addition, three key miRNAs (miR-98-5p, miR-7-5p, and miR-182-5p) and six key genes (murine double minute 2, MDM2; glycogen synthase kinase 3-beta, GSK3B; transmembrane P24-trafficking protein 10, TMED10; DDB1 and CUL4-associated factor 12, DCAF12; caspase 3, CASP3; and ring finger protein 44, RNF44) were screened, among which the miR-7-5p → TMED10/DCAF12, miR-98-5p → CASP3/RNF44, and miR-182-5p → GSK3B pairs were observed in the regulatory network. Moreover, the expression levels of TMED10, miR-7-5p, CASP3, miR-98-5p, GSK3B, and miR-182-5p showed a negative correlation with qRT-PCR verification.ConclusionMiR-98-5p, miR-7-5p, miR-182-5p, MDM2, GSK3B, TMED10, DCAF12, CASP3, and RNF44 may play critical roles in OA progression.

Global Histone H3 Lysine 4 Trimethylation (H3K4me3) Landscape Changes in Response to TGFβ.

TGFβ expression acts as a biomarker of poor prognosis in prostate cancer. It plays a dual functional role in prostate cancer. In the early stages of the tumor, it acts as a tumor suppressor while at the later stages of tumor development, it promotes metastasis. The molecular mechanisms of action of TGFβ are largely understood through the canonical and non-canonical signal transduction pathways. Our understanding of the mechanisms that establish transient TGFβ stimulation into stable gene expression patterns remains incomplete. Epigenetic marks like histone H3 modifications are directly linked with gene expression and they play an important role in tumorigenesis. In this report, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) to identify the genome-wide regions that undergo changes in histone H3 Lysine 4 trimethylation (H3K4me3) occupancy in response to TGFβ stimulation. We also show that TGFβ stimulation can induce acute epigenetic changes through the modulation of H3K4me3 signals at genes belonging to special functional categories in prostate cancer. TGFβ induces the H3K4me3 on its own ligands like TGFβ, GDF1, INHBB, GDF3, GDF6, BMP5 suggesting a positive feedback loop. The majority of genes were found to be involved in the positive regulation of transcription from the RNA polymerase II promoter in response to TGFβ. Other functional categories were intracellular protein transport, brain development, EMT, angiogenesis, antigen processing, antigen presentation via MHC class II, lipid transport, embryo development, histone H4 acetylation, positive regulation of cell cycle arrest, and genes involved in mitotic G2 DNA damage checkpoints. Our results link TGFβ stimulation to acute changes in gene expression through an epigenetic mechanism. These findings have broader implications on epigenetic bases of acute gene expression changes caused by growth factor stimulation.

Endoplasmic reticulum-targeting nanomedicines for cancer therapy

In drug development research, the specific affinity of drug delivery systems for particular pathological sites, cells, and even subcellular structures can largely reduce the side effects, systemic toxicity, and minimally required drug dose. Since endoplasmic reticulum (ER) plays an essential role in many important cellular activities and functions, including the synthesis and intracellular transport of secretory and membrane-associated proteins, the synthesis and transport of steroids and lipids, Ca2+ signaling, and stress response, the ER dysfunction is associated with many diseases. Developing delivery systems that can effectively enter the cell and target ER is highly important for achieving satisfactory therapeutic performance in certain diseases, such as cancer. This review summarizes the currently available ER-targeting nanoparticle systems for cancer therapy. We first discuss the background knowledge involved in the ER-targeted nanotherapies for cancer. Then, we describe the examples about ER-targetable nanoparticles from several aspects, such as the components, the construction, and the therapeutic effect and mechanisms of these nanoparticles. Finally, we summarize the ER-targeted nanotherapeutic strategies for cancer and point out the current challenges and future research directions in this field. We hope that this review will promote the further development of ER-targeting nanotheranostics and inspire future research on ER-targeted cancer treatments.