The microRNA159 (miR159) family represses the conserved GAMYB-like genes that encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) signaling in anthers and germinating seeds. In Arabidopsis (Arabidopsis thaliana), the two major miR159 family members, miR159a and miR159b, are functionally specific for two GAMYB-like genes, MYB33 and MYB65. These transcription factors have been shown to be involved in anther development, but there are differing reports about their role in the promotion of flowering and little is known about their function in seed germination. To understand the function of this pathway, we identified the genes and processes controlled by these GAMYB-like genes. First, we demonstrate that miR159 completely represses MYB33 and MYB65 in vegetative tissues. We show that GA does not release this repression and that these transcription factors are not required for flowering or growth. By contrast, in the absence of miR159, the deregulation of MYB33 and MYB65 in vegetative tissues up-regulates genes that are highly expressed in the aleurone and GA induced during seed germination. Confirming that these genes are GAMYB-like regulated, their expression was reduced in myb33.myb65.myb101 seeds. Aleurone vacuolation, a GA-mediated programmed cell death process required for germination, was impaired in these seeds. Finally, the deregulation of MYB33 and MYB65 in vegetative tissues inhibits growth by reducing cell proliferation. Therefore, we conclude that miR159 acts as a molecular switch, only permitting the expression of GAMYB-like genes in anthers and seeds. In seeds, these transcription factors participate in GA-induced pathways required for aleurone development and death.
Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).
Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.
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