Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease-Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock-down of endocytosis gene Rvs161 improved the concentration of α-amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock-down mutants in Rvs161 and End3 genes with 2-fold and 3-fold α-amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae. This result is likely generalizable to other eukaryotic secretion hosts.
3853 B-cell differentiation is tightly regulated by synchronized suppression and/or induction of specific transcription factors. Among them, B-cell lymphoma 6 (BCL6) and PRDM1 are considered to be master regulators for germinal center formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 also have been associated with lymphomagenesis. Their regulation still need further study especially at the posttranscriptional level. Here, by using co-culture system and whole genomic microRNA microarray profiling, we show for the first time that direct B lymphoma cell-stroma cell contact between follicular dendritic cells and B-lymphocytes could induce upregulation of miR-30 family and downregulation of miR-9 and let-7 family. In silico analysis showed that miR-30s can target genes BCL6 and miR-9/let7 can target PRDM1 with direct binding sites in 3`UTR region of their mRNAs. The microarray data can be proved by microRNA specific Q-RT-PCR. Specifically, by both gain of function and loss of function studies, we functionally verified that FDCs Regulate Expression of BCL6 and PRDM1 via Cell-Cell Direct Contact induced correlated microRNA dysregulation. To further validate the direct interaction between BCL6 and miR-30, we constructed luciferase reporters containing the BCL6 3`-UTR that included miR-30 binding sites and a mutant 3`-UTR harboring mutations in the “seed pairing” sequences of the miR-30 binding site. Co-transfection of miR-30 and reporter construct into cells significantly decreased luciferase activity in wild-type but not in mutant BCL6-3`-UTR transfected cells, supporting the role of miR-30 family in the regulation of BCL6 expression. BCL6 and PRDM1 and their regulation miRNAs, let-7 and miR-30, also can be validated in primary normal B-lymphocytes and lymphoma cells by using our co-culture system. Dysregulation of BCL6 and PRDM1 is often associated with lymphomagenesis. We firstly identified that BCL6 is the direct target of miR-30 family and also verified PRDM1 is the target of miR-9, and let-7 in our system. Our studies provide a novel mechanism of post-transcriptional regulation of BCL6 and PRDM1 by several microRNAs. In the context of micro-environment, it provides a clue for germinal center B-cell differentiation as well as B-cell lymphomas progression regulated by lymphocyte cell-stroma cell contact through microRNAs. Disclosures: No relevant conflicts of interest to declare.
3122 Adult T-cell leukemia/lymphoma (ATL) is an aggressive, fatal malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type. ATL has been classified into four main subtypes, indolent smoldering, chronic forms, acute and lymphomatous forms. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. Compelling evidence supports a pivotal role for altered tumor suppressor genes involved in leukemogenesis and lymphomagenesis of ATL as well as progression of the disease from the chronic/smoldering to the acute/lymphomatous type. Further investigations are needed as to how the alteration of cell cycle regulatory genes are involved in the aberrant growth of HTLV-infected T cells. The emerging role of microRNAs in tumor development and viral infection has prompted investigations on the role of microRNA in ATL pathogenesis and progression. We therefore performed microRNA microarrays on 16 lymphomatous tissues of ATL patients and normal CD4 lymphocytes of 5 healthy donors. After normalization, we identified a set of aberrantly expressed miRNAs in ATL lymphoma samples when compared with control CD4 lymphocytes including 49 upregulated microRNAs and 89 downregulated microRNAs. MiR-150, miR-24, miR-221, miR-222, miR-142-5p, miR-92b, miR-93, miR-181a, miR-103, miR-107 stands out as highly elevated miRNAs in ATL samples while miR-30b, miR-34a, miR-20b, miR-196b, miR-361-5p, miR-520b/e are found downregulated in ATLL samples. Using gain-and-loss experimentations, we demonstrated miR-181a overexpression decreases, whereas miR-181a inhibition increases a proapoptotic protein, Bim, levels by directly targeting Bim. Furthermore computational analysis, and previous studies revealed that that miR-214, miR-22, miR-93 increased AKT activity by repressing PTEN expression, and miR-221/222 and miR-93 promoted cell proliferation through inhibition of CDKN1B (p27). Overall, this study demonstrated a distinct set of miRNAs that are known to be critical in cellular transformation, representing a novel molecular mechanism and potential therapeutic targets in ATL lymphoma. Disclosures: No relevant conflicts of interest to declare.
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