1,721,024 research outputs found
Deciphering the Role of the Classical Protein Secretion Pathway in the Translocation of HSPA1A to the Cell Surface in Stressed and Cancer Cells
Full text linkHSPA1A, is a 70-kDa molecular chaperone, which is overproduced in stressed and cancer cells. HSPA1A maintains the cell's dynamic balance and promotes cell survival. Additionally, HSPA1A moves to the cell surface and the extracellular medium of cancer and stressed cells. Cell surface and extracellular HSPA1A promote tumor resistance and alter immune function making this protein an attractive target for new therapeutics. However, how the protein translocates to the cell surface and transports to the extracellular medium remain largely undefined because HSPA1A does not contain known translocation signals. The purpose of this project is to determine the importance of the classical protein secretion pathway by inhibiting the first steps of this pathway. To this end, cells were treated with two compounds that both disrupt the endoplasmic reticulum (ER) to Golgi protein transfer using different mechanisms, and after heat-shock cell surface HSPA1A was quantified using confocal microscopy. Preliminary results using both compounds suggest that HSPA1A's cell surface translocation is independent of the ER-Golgi pathway. These results, which are currently being finalized and controlled, provide important first clues as to the mechanism of HSPA1A's translocation in stressed and cancer cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Interaction of HSPA1A, A70-Kda Molecular Chaperone, With Phosphatidylinositol 4-Phosphate [PI(4)P] in Stressed Mammalian Cells
Full text linkThe translocation of HSPA1A, a molecular chaperone, to the plasma membrane (PM) of tumor cells facilitates resistance to radiation therapy and the formation of distant metastases. Inhibition of HSPA1A's PM localization is a promising therapeutic, though it is unknown how this protein is recruited to the PM as it does not contain any known translocation signals or lipid-binding sites. Yet, HSPA1A binds to anionic lipids such as phosphatidylserine (PS) and monophosphorylated phosphatidylinositides (PIPs) in vitro and the binding to intracellular PS enables the PM localization of the chaperone. I hypothesized that HSPA1A's PM localization depends on its interaction with PIPs and that the lipid-binding and chaperone functions are independent. To test these hypotheses, I selectively depleted cellular lipids by using either lipid-biosensors or drugs that manipulate lipid metabolizing enzymes. Furthermore, I determined the effect of lipids on HSPA1A's structure and chaperone activities. My data strongly support the notion that HSPA1A's PM localization is lipid- driven as depletion of specific PIPs resulted in a loss of PM-localized HSPA1A. Furthermore, these results corroborate that lipid-binding does not significantly affect the protein's ability to perform its chaperone functions, implying that the sites that mediate the two functions are distinct. Collectively, this knowledge provides the basis for future interventions to specifically target the lipid-binding function of HspA1A as opposed to targeting the chaperone function, which has detrimental side effects
Characterization of the Relationship Between Gene Expression and Lipid Composition in Heat-Stressed HeLa Cells
No full textThe cellular stress response (CSR) is a highly conserved biological toolbox targeting abiotic and biotic threats to survival. A subset of the CSR is the heat-stress response (HSR), classically exemplified by molecular chaperone proteins in all living cells. The HSR has previously been characterized via the molecular arsenal of genomics, transcriptomics, metabolomics, and proteomics; however, multi-omic integrative analyses are lacking. Furthermore, a leading model of the dynamic plasma membrane implies that its lipid-protein composition is a regulator of HSR activation. Given that the mammalian HSR involves differential expression of far more genes than solely the chaperones and their inducers, I hypothesize heat-shock (HS) induces the transcription of genes encoding lipid metabolizing enzymes, causing an increase in de novo lipogenesis in HeLa cells. To this end, HeLa cells were subjected to one of three experimental conditions: control, HS, or HS with eight hours of recovery at control conditions. Mass spectrometry was used to generate a lipidome for each condition and mRNA-Sequencing a transcriptome for each condition. The HS lipidome demonstrated evidence of significant increases in fatty acids (FA), glycerophospholipids, and sphingolipids with pathway level enrichment of biosynthesis of FA. In addition to over 1800 differentially expressed genes (DEG), the transcriptome demonstrated marginal but present evidence for lipid metabolism in both functional enrichment and GO analyses. Joint-pathway analyses determined that linolenic acid metabolism, sphingolipid metabolism, and FA metabolism pathways were significantly enriched. Network analyses identified cholesterol import genes of interest. Seven proposed DEG-based mechanisms evidence a successful multi-omics integration that enables future contributions to approximate a systems-level model of the relationship between transcriptome and lipidome in any stress context.https://doi.org/10.5281/zenodo.740702
Transcriptomic Analysis of Zinc Transporters in Mliv Mouse Model
Full text linkZinc (Zn2+) plays key roles in many diverse cellular processes but that its levels must be highly regulated to maintain healthy brain function. Zinc dyshomeostasis and the loss of the TRPML1 channel has been associated with neurodegenerative diseases such as Mucolipidosis type IV (MLIV). There are distinct proteins that maintain cellular zinc levels, the SLC30A family (ZNT 1-10; involved in Zn2+ efflux), and the SLC39A family (ZIP 1-14; involved in Zn2+ influx). Recently, TMEM163 was characterized as a Zn2+ efflux protein, which happens to interact with the TRPML1 channel and has been proposed to influence brain zinc levels. A previous report suggests that zinc transporter dysfunction affects brain Zn2+ homeostasis and contributes to MLIV. In MLIV Mcoln1–/– knockout (KO) mouse model, motor deficit is linked with disease pathology in the cerebellum. The current study aims to determine changes in the expression patterns of zinc transporters in the mouse cerebellum at three developmental ages to potentially explain the role they play in MLIV pathology. RNA-seq and qPCR confirmed that in the 7-month-old Mcoln1–/– KO mice, Znt1, Znt4, and Zip1 genes were significantly downregulated. At 3-months old, Zip1 remained significantly upregulated, Znt4 and Znt9 were significantly downregulated, and Znt1 was upregulated. At 1-month, only Znt4 was significantly downregulated. In conclusion, abnormal transcript levels of zinc transporters in the cerebellum of MLIV mouse model are associated with the disease process. By understanding the expression patterns of brain zinc transporters, we can further study possible mechanisms underlying the pathology of MLIV disease.https://doi.org/10.5281/zenodo.656811
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Delineating the Secretory Pathway of HSPA1A After Heat-Shock
Full text linkExtracellular HSPA1A (eHSPA1A), the stressed-induced member of the HSP70 molecular chaperone family, regulates many vital processes, as it activates the innate and adaptive immune responses and promotes tumor cell death and suppression. Additionally, membrane-bound HSPA1A marks several types of cancer cells, making them resistant to therapy and radiation. Although HSPA1A translocates and anchors at the plasma membrane (PM) and is actively secreted from viable cells, it lacks known transmembrane domains, lipid-binding domains, and signal peptides. However, the relationship between membrane localization and secretion of HSPA1A remains largely undefined. Clarifying this relationship is vital for understanding HSPA1A's function in tumor progression and metastasis. I hypothesized that the lipid-driven translocation of HSPA1A to the PM is the intermediate step of its secretion to the extracellular medium. To test my hypothesis, I monitored the secretion of HSPA1A after heat-shock in the presence and absence of specific lipid biosensors that will manipulate different membrane phospholipids. My results show that eHSPA1A increases continuously during the cells' recovery after mild non-apoptotic heat shock. Furthermore, eHSPA1A is found exclusively in extracellular vesicles (EVs) under these conditions. Additionally, PM localized HSPA1A increased during recovery from heat shock, reached a maximal at 8 hours post-stress, and then declined. Depleting a specific lipid target via co-transfection with lipid biosensors resulted in inhibition of HSPA1A's PM localization but did not affect the EM export of the protein via EVs. Both the timing and lipid-depletion support the notion that HSPA1A's PM localization and EM export are independent and probably sequential events. My research provides new information on the mechanism of HSPA1A's secretion and its relationship to lipid-binding, which will be used in future research to define the mechanistic details and determine what recruits HSPA1A for EM export in a form that can activate the immune system against tumor cells.https://doi.org/10.5281/zenodo.656152
Identification of Signaling Pathways Associated With Activation of the Mechanosensitive Channel, TcMscS in Trypanosoma cruzi
Full text linkChagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. With no successful treatment options, there is an urgent need to find new therapeutic approaches for this disease. The protozoan Trypanosoma cruzi faces environmental challenges to maintain homeostasis and complete its lifecycle. A bacterial-like mechanosensitive channel TcMscS, found in the parasites, plays a vital role in growth, infectivity, differentiation, and osmoregulation. However, the information available on the signaling pathways responsible for these phenotypes is scarce. This study attempted to identify the signaling pathways downstream of TcMscS activation. Based on RNA-Sequence analysis of TcMscS KO mutants, diacylglycerol kinase (DAGK) and kinesin were selected as candidate genes for this study, and their role was investigated. Localization studies revealed the possible localization of kinesin near the contractile vacuole and the flagellar pocket. Using CRISPR/Cas9 methods, the expression level of the candidate genes was manipulated. The candidate gene KO mutants showed significant growth defects and cytokinesis defects. The kinesin KO mutants showed abnormal motility and a bent flagellum. Overall, this study reveals the possible role of diacylglycerol kinase and kinesin in parasite growth and cell division.https://doi.org/10.5281/zenodo.656220
Identifying and Examining the Role of Chemical Modifications in the Differential Splicing Activities of Two Related RNA Binding Proteins
Full text linkRNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N- terminal, Linker 1, and Linker 2 regions. In addition, we tested the RNA binding affinity of these modified proteins via band shift assays. Our results highlight that extract incubated PTBPs have different RNA binding affinities to an RNA sequence containing high affinity PTPB binding sites. We find that extract incubated PTBP1 and PTBP2 differ in their ability to form a higher order oligomeric complex that is required for efficient splicing repression. Collectively, our findings suggest a role for PTBP phosphorylation in higher order oligomeric complex formation
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