ASSEMBLY AND DEGRADATION OF A TRIMERIC MEMBRANE PROTEIN ACRB

Multidrug efflux pumps are membrane proteins that actively transport foreign objects out of cells. The active efflux of these pumps is a critical self-defense mechanism that enables the survival of bacteria under hostile environments. Efflux pump AcrB is a member of the Resistance-Nodulation-Division (RND) super family. In E. coli, it associates with periplasmic protein AcrA and outer membrane channel TolC to extrude a variety of noxious compounds out of cell from both the cytoplasm and the periplasm. My dissertation research focused on two aspects of this multidrug efflux pump: the oligomerization process during the biogenesis of AcrB and its degradation. Oligomerization is an important aspect of the structure and function for many proteins and has been the subject of many studies. However, most of such studies focused on soluble proteins. The oligomerization process of membrane proteins, including AcrB, is rarely explored. In chapter 2, the co-assembly of AcrB variants co-expressed in the same cell was used as a tool to investigate the assembly of AcrB trimers during its bio-production. By monitoring the portion of pure trimers containing only one type of subunit and hybrid trimers containing a mixture of the two kinds of subunits, it was found that the oligomerization of membrane proteins is not a random process as the formation of pure trimer is favored. In chapter 3, the GALLEX system was used to monitor AcrB oligomerization in cells under the native condition. Previously GALLEX has only been used to monitor the oligomerization of small transmembrane proteins. By constructing a series of fusion proteins with different linker length between LexA and AcrB, and optimizing inducer concentration, we finally developed a system that could be used to differentiate AcrB trimers of different oligomerization affinities. While chapters 2 and 3 focus on the trimerization of AcrB, a critical step of its biogenesis, chapters 4 and 5 focus on its life time and degradation. In chapter 4, the life time of AcrB was measured by incorporating non-natural amino acid azidohomoalanine (AHA) into protein translation. Using this method, it was determined that that the half-life of both AcrA and AcrB in E. coli were six days. The surprisingly long lifetime of these detoxification proteins might represent a strategy by the bacteria to conserve energy and maximize their competition niche for survival in a hostile environment. In chapter 5, the degradation process of ssra tagged AcrB was investigated. In-vivo degradation test showed that properly inserted AcrB can be digested after addition of ssra-tag to its C-terminus. It was found that cytoplasmic unfoldase-protease complex ClpXP and chaperone SspB are involved in the degradation. In vitro assay is still being optimized to quantitatively analyze the activity of ClpXP in the degradation of AcrB

Longitudinal Optogenetic Motor Mapping Revealed Structural and Functional Impairments and Enhanced Corticorubral Projection after Contusive Spinal Cord Injury in Mice

Current evaluation of impairment and repair after spinal cord injury (SCI) is largely dependent on behavioral assessment and histological analysis of injured tissue and pathways. Here, we evaluated whether transcranial optogenetic mapping of motor cortex could reflect longitudinal structural and functional damage and recovery after SCI. In Thy1-Channelrhodopsin2 transgenic mice, repeated motor mappings were made by recording optogenetically evoked electromyograms (EMGs) of a hindlimb at baseline and 1 day and 2, 4, and 6 weeks after mild, moderate, and severe spinal cord contusion. Injuries caused initial decreases in EMG amplitude, losses of motor map, and subsequent partial recoveries, all of which corresponded to injury severity. Reductions in map size were positively correlated with motor performance, as measured by Basso Mouse Scale, rota-rod, and grid walk tests, at different time points, as well as with lesion area at spinal cord epicenter at 6 weeks post-SCI. Retrograde tracing with Fluoro-Gold showed decreased numbers of cortico- and rubrospinal neurons, with the latter being negatively correlated with motor map size. Combined retro- and anterograde tracing and immunostaining revealed more neurons activated in red nucleus by cortical stimulation and enhanced corticorubral axons and synapses in red nucleus after SCI. Electrophysiological recordings showed lower threshold and higher amplitude of corticorubral synaptic response after SCI. We conclude that transcranial optogenetic motor mapping is sensitive and efficient for longitudinal evaluation of impairment and plasticity of SCI, and that spinal cord contusion induces stronger anatomical and functional corticorubral connection that may contribute to spontaneous recovery of motor function

Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

<p>Abstract</p> <p>Background</p> <p>Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells.</p> <p>Results</p> <p>Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In addition, chromatin immunoprecipitation assay further confirmed the results of reporter gene studies by showing the specific binding of NFAT2 to the -201 to +71 region.</p> <p>Conclusion</p> <p>In the present study, we demonstrated that NFAT2 directly stimulates transcription of FasL in high level CORT-treated mLTC-1. In conclusion, the present study provides further evidence for our finding that CORT-induced FasL expression in Leydig cells is mediated by NFAT.</p

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB

Hyperglycemia in apolipoprotein E-deficient mouse strains with different atherosclerosis susceptibility

<p>Abstract</p> <p>Background</p> <p>Type 2 diabetes mellitus (T2DM) is associated with an increased risk of atherosclerotic vascular disease, but it is unknown whether the other way around is true too. C57BL/6 (B6) and BALB/cJ (BALB) are two mouse strains that differ markedly in their susceptibility to atherosclerosis. In this study we investigated the development of diet-induced T2DM in these two strains.</p> <p>Methods and Results</p> <p>When deficient in apolipoprotein E (apoE^-/-) and fed a Western diet for 12 weeks, atherosclerosis-susceptible B6 mice developed significant hyperglycemia. In contrast, atherosclerosis-resistant BALB apoE^-/-mice had much lower plasma glucose levels than B6.apoE^-/-mice on either chow or Western diet and during an intraperitoneal glucose tolerance test. In response to glucose BALB.apoE^-/-mice displayed both the first and second phases of insulin secretion but the second phase of insulin secretion was absent in B6.apoE^-/-mice. In response to insulin B6.apoE^-/-mice showed a deeper and longer-lasting fall in blood glucose levels while BALB.apoE^-/-mice showed little reduction in glucose levels. Pancreatic islet area of BALB.apoE^-/-mice on light microscopy nearly doubled the area of B6.apoE^-/-mice. Most circulating proinflammatory cytokines were lower in BALB.apoE^-/-than in B6.apoE^-/-mice on the Western diet, as determined by protein arrays. Increased macrophage infiltration in islets was observed in B6.apoE^-/-mice by immunostaining for Mac2 and also by flow cytometry.</p> <p>Conclusion</p> <p>This study demonstrates that defects in insulin secretion rather than defects in insulin resistance explain the marketed difference in susceptibility to T2DM in the B6.apoE^-/-and BALB.apoE^-/-mouse model. A smaller islet mass and more prominent islet inflammation may explain the vulnerability of B6.apoE^-/-mice to diet-induced diabetes.</p

Metaverse : concept, key technologies, and vision

Metaverse is a collective term for all economic and social activities in the space where the physical world, digital world, and consciousness world are interactively integrated and mutually empowered. The metaverse is the advanced stage of digital civilization and the future formation of human society. The basis for developing metaverse is general digital technologies such as high-performance network, high-performance storage, high-performance computing, high-performance security, and artificial intelligence. On the basis of the above, the key to developing the metaverse lies in researching core technologies such as digital life technologies, trusted collaborative network technologies, natural interaction technologies, ubiquitous operating system technologies, technologies and methods for computational experiments, and theories and technologies for crowd intelligence science. We should take typical metaverse application scenarios as entry points, such as the key fields of agriculture, industry, service industry, military, social governance, and other economic and social areas, to break through key metaverse technologies and implement pilot demonstration projects of metaverse. Through the demonstration, we can systematically promote the application of metaverse in economy and society from point to line, and continuously iterate and evolve the metaverse technology to advance the metaverse to a higher stage. This paper systematically analyzes the current development status and future directions of metaverse from the concept, key technology, and vision of metaverse, paving the way for the subsequent research of metaverse

Precise Measurement of Gravity Variations During a Total Solar Eclipse

The variations of gravity were measured with a high precision LaCoste-Romberg D gravimeter during a total solar eclipse to investigate the effect of solar eclipse on the gravitational field. The observed anomaly $(7.0 \pm 2.7) \times 10^{-8}$ m/s$^2$ during the eclipse implies that there may be a shielding property of gravitation