Se-Hoon Sim

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.

Escherichia coli ribonuclease III activity is downregulated by osmotic stress: consequences for the degradation of bdm mRNA in biofilm formation

During the course of experiments aimed at identifying genes with ribonuclease III (RNase III)‐dependent expression in Escherichia coli, we found that steady state levels of bdm mRNA were dependent on cellular concentrations of RNase III. The half‐lives of adventitiously overexpressed bdm mRNA and the activities of a transcriptional bdm‘–’cat fusion were observed to be dependent on cellular concentrations of RNase III, indicating the existence of cis‐acting elements in bdm mRNA responsive to RNase III. In vitro and in vivo cleavage analyses of bdm mRNA identified two RNase III cleavage motifs, one in the 5′‐untranslated region and the other in the coding region of bdm mRNA, and indicated that RNase III cleavages in the coding region constitute a rate‐determining step for bdm mRNA degradation. We also discovered that downregulation of the ribonucleolytic activity of RNase III is required for the sustained elevation of RcsB‐induced bdm mRNA levels during osmotic stress and that cells overexpressing bdm form biofilms more efficiently. These findings indicate that the Rcs signalling system has an additional regulatory pathway that functions to modulate bdm expression and consequently, adapt E. coli cells to osmotic stress.

Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the alpha-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the alpha-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.

MCL-1ES, a novel variant of MCL-1, associates with MCL-1L and induces mitochondrial cell death

MINT‐7255756:MCL1‐ES (uniprotkb:Q07820‐2) physically interacts (MI:0914) with COX IV (uniprotkb:P13073) by anti tag coimmunoprecipitation (MI:0007)

Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter

MacB is a noncanonic ABC-type transporter within Gram-negative bacteria, which is responsible both for the efflux of macrolide antibiotics and for the secretion of heat-stable enterotoxin II. In Escherichia coli, MacB requires the membrane fusion protein MacA and the multifunctional outer membrane channel TolC to pump substrates to the external medium. Sequence analysis of MacB suggested that MacB has a relatively large periplasmic region. To gain insight into how MacB assembles with MacA and TolC, we determined the crystal structure of the periplasmic region of Actinobacillus actinomycetemcomitans MacB. Fold matching program reveals that parts of the MacB periplasmic region have structural motifs in common with the RND-type transporter AcrB. Since it behaved as a monomer in solution, our finding is consistent with the dimeric nature of full-length MacB, providing an insight into the assembly in the tripartite efflux pump.

The tip region of the MacA α-hairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump

The tripartite efflux pump MacAB-TolC found in gram-negative bacteria is involved in resistance to antibiotics. We previously reported the funnel-like hexameric structure of the adaptor protein MacA to be physiologically relevant. In this study, we investigated the role of the tip region of its alpha-hairpin, which forms a cogwheel structure in the funnel-like shape of the MacA hexamer. Mutational and biochemical analyses revealed that the conserved residues located at the tip region of the alpha-hairpin of MacA play an essential role in the binding of TolC. Our findings offer a molecular basis for understanding the drug resistance of pathogenic bacteria.

HIP1R interacts with a member of Bcl-2 family, BCL2L10, and induces BAK-dependent cell death.

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. BCL2L10 (human Diva or BCL-B) is a member of the Bcl-2 family that has contradictory functions in apoptosis. In the present study, we identified the Huntington-interacting protein 1-related (HIP1R) protein following a search for Diva-interacting proteins using the yeast two-hybrid system. HIP1R is a multi-domain protein that regulates the clathrin-mediated endocytic machinery and actin assembly in cells. Interaction of endogenous proteins of BCL2L10 and HIP1R in 293T cells was determined by immunoprecipitation, and their direct association was confirmed by the Far-Western analysis. The deletion of both the AP180-homology (ANTH) and F-actin-binding the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domains of HIP1R greatly compromised its binding ability to BCL2L10. Ectopic expression of HIP1R resulted in moderate cell death of 293T cells in conjunction with the dissipation of mitochondrial membrane potential and caspase 9 activation. A member of proapoptotic Bcl-2 family, BAK, was required for HIP1R to induce cell death, while BAX was dispensable. In addition, BCL2L10 was associated with endogenous caspase 9, and their binding was augmented by HIP1R overexpression. Thus, this study provided the previously unknown function of HIP1R involved in the intrinsic cell death pathway and further explored possible mechanisms by which HIP1R induces cell death.

Effects of Escherichia coli RraA Orthologs of Vibrio vulnificus on the Ribonucleolytic Activity of RNase E In Vivo

RraA is a recently discovered protein inhibitor of RNase E that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. In the genome of Vibrio vulnificus, two open reading frames that potentially encode proteins homologous to E. coli, RraA-designated RraAV1 and RraAV2, have respectively 80.1% and 59.0% amino acid identity to RraA. The authors report that coexpression of RraAV1 protein in E. coli cells overproducing RNase E rescued these cells from growth arrest and restored their normal growth, whereas coexpression of RraAV2 protein further inhibited the growth of E. coli cells, whose growth was already impaired by overproduction of RNase E. Analyses of the steady-state level of various RNase E substrates indicated that the coexpression of RraAV1 more efficiently inhibited RNase E action than coexpression of RraA, and consequently resulted in the more increased abundance of each RNA species tested in vivo. The inhibitory effect by RraAV2 coexpression on RNase E was observed only in the case of trpA mRNA, indicating the possibility of RNA substrate-dependent inhibition of RraAV2 on RNase E. The findings suggest that these regulators of ribonuclease activity have both a conserved inhibitory function and a differential inhibitory activity on RNase E-like enzymes across the species.

Increased expression of the testicular estrogen receptor alpha in adult mice exposed to low doses of methiocarb.

Methiocarb is a widely used carbamate pesticide and a suspected endocrine disrupter. The objective of this study was to examine the in vivo effects of methiocarb at low doses on testicular expression of steroid receptors, spermatogenesis and sperm quality in adult mice. Eighteen‐week‐old DBA/2 males were treated with daily intraperitoneal injection of methiocarb (0, 0.03, 0.3, 1.0 or 3.0 µg kg−1 of body weight) for 20 days. Kidney and liver weights were significantly increased in the 1.0 or 3.0 µg kg−1 treatment groups (P < 0.05). The testicular expression of estrogen receptor α (ERα) was significantly increased in mice treated with methiocarb as confirmed by Western blot analysis. The sperm production and sperm quality of the methiocarb‐exposed mice were not significantly altered as determined using a computer‐assisted sperm analysis system. Therefore, these results demonstrate, that although the exposure to methiocarb at low doses alters testicular ERα expression in adult mice, both sperm production and quality remain unaffected. Copyright

Two Tandem RNase III Cleavage Sites Determine betT mRNA Stability in Response to Osmotic Stress in Escherichia coli

While identifying genes regulated by ribonuclease III (RNase III) in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.