Identification of the Transcriptional Regulator NcrB in the Nickel Resistance Determinant of Leptospirillum ferriphilum UBK03

The nickel resistance determinant ncrABCY was identified in Leptospirillum ferriphilum UBK03. Within this operon, ncrA and ncrC encode two membrane proteins that form an efflux system, and ncrB encodes NcrB, which belongs to an uncharacterized family (DUF156) of proteins. How this determinant is regulated remains unknown. Our data indicate that expression of the nickel resistance determinant is induced by nickel. The promoter of ncrA, designated pncrA, was cloned into the promoter probe vector pPR9TT, and co-transformed with either a wild-type or mutant nickel resistance determinant. The results revealed that ncrB encoded a transcriptional regulator that could regulate the expression of ncrA, ncrB, and ncrC. A GC-rich inverted repeat sequence was identified in the promoter pncrA. Electrophoretic mobility shift assays (EMSAs) and footprinting assays showed that purified NcrB could specifically bind to the inverted repeat sequence of pncrA in vitro; this was confirmed by bacterial one-hybrid analysis. Moreover, this binding was inhibited in the presence of nickel ions. Thus, we classified NcrB as a transcriptional regulator that recognizes the inverted repeat sequence binding motif to regulate the expression of the key nickel resistance gene, ncrA

TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status

Melville Trust for the Care and Cure of Cancer to SP and SS.Background: TGF beta has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21(Cip1) and pRB, three critical regulators of the G1/S transition are variably involved in TGF beta-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGF beta-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated. Methods: Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGF beta for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleavedcapsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR. Results: We found that TGF beta similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGF beta. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMA alpha and Vimentin) studied remained unchanged. TGF beta induced high levels of apoptosis in p53-/-, Rb-/-, p21(cip1)-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21(cip1)were less sensitive to TGF beta-induced apoptosis. Conclusion: Although p53, p21(Cip1) and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGF beta-driven inhibition of hepatocytes proliferation, but only slightly modulate TGF beta-induced apoptosis. This effect may depend on other parameters such as proliferation and the presence of other regulatory proteins as suggested by the consequences of p53, p21(Cip1) double deficiency. Similarly, p53, p21(Cip1) and pRB deficiency had no effect on the morphological changes and loss of cell adhesion which is thought to be critical for metastasis. This indicates that possible association of these genes with metastasis potential would be unlikely to involve TGF beta-induced EMT.Publisher PDFPeer reviewe

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activat

One-way membrane trafficking of SOS in receptor-triggered Ras activation

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multipl

A tight tunable range for Ni(II) sensing and buffering in cells

The metal affinities of metal-sensing transcriptional regulators co-vary with cellular metal concentrations over more than 12 orders of magnitude. To understand the cause of this relationship, we determined the structure of the Ni(II) sensor InrS and then created cyanobacteria (Synechocystis PCC 6803) in which transcription of genes encoding a Ni(II) exporter and a Ni(II) importer were controlled by InrS variants with weaker Ni(II) affinities. Variant strains were sensitive to elevated nickel and contained more nickel, but the increase was small compared with the change in Ni(II) affinity. All of the variant sensors retained the allosteric mechanism that inhibits DNA binding following metal binding, but a response to nickel in vivo was observed only when the sensitivity was set to respond in a relatively narrow (less than two orders of magnitude) range of nickel concentrations. Thus, the Ni(II) affinity of InrS is attuned to cellular metal concentrations rather than the converse

One-way membrane trafficking of SOS in receptor-triggered Ras activation

SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane-recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2:SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted membrane experiments, these Grb2-independent interactions are sufficient to retain SOS on the membrane for many minutes, during which a single SOS molecule can processively activate thousands of Ras molecules. These observations raise questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative reconstituted SOS-deficient chicken B cell signaling systems combined with single molecule measurements in supported membranes. These studies reveal an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until it is actively removed via endocytosis