Inhibition Of Division Research Articles

SosA inhibits cell division in Staphylococcus aureus in response to DNA damage.

SummaryInhibition of cell division is critical for viability under DNA‐damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS‐induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C‐terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild‐type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C‐terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.

A novel hiPSC-derived system for hematoendothelial and myeloid blood toxicity screens identifies compounds promoting and inhibiting endothelial-to-hematopoietic transition

The exposure to toxic environmental and pharmaceutical substances can pose a long-term risk to human's health. In this study, we sought to investigate the potential of our recently developed method for induction of myeloid hematoendothelial and blood cells by overexpression of two transcription factors, GATA2 and ETV2, in human induced pluripotent stem cells (hiPSCs) for toxicity screening. For the primary screen in a high-throughput format, we selected twenty-two chemicals with various degrees of cytotoxicity available from the NIEHS National Toxicology Program (Tox21). The compounds were applied during the endothelial-to-hematopoietic transition and to differentiated myeloid progenitors growing in suspension. The system was capable of identifying compounds with both inhibitory and favorable effects on hematopoietic network, changes in expression of hematopoietic markers, and mitochondrial and cytotoxicity. The findings were confirmed and further investigated by secondary screens, colony forming cell assay, and gene expression profiling. The hematoendothelial toxicity of 5-fluorouracil, berberine chloride, and benzo(a)pyrene is characterized by the inhibition of cell division and a shift of hematopoietic programming to non-hemogenic endothelial and mesenchymal fates. This study demonstrates the feasibility of transcription factor (TF)-based differentiation systems to monitor endothelial and hematotoxicity and serves as an informative platform for screening myelosuppressive or stimulatory drugs and mechanistic studies of their action.

Spatial Dependence of Stimulus Competition in the Avian Nucleus Isthmi Pars Magnocellularis

The nucleus isthmi pars magnocellularis (Imc) is a group of specialized inhibitory neurons in the midbrain tegmentum, thought to be conserved across vertebrate classes. Past anatomical work in reptiles has suggested a role for it in stimulus selection, which has been supported by recent studies in avians. Additionally, focal inactivation of Imc neurons is known to abolish all competitive interactions in the optic tectum (OT; SC in mammals), a midbrain sensorimotor hub that is critical for the control of spatial attention, thereby revealing a key role for Imc in stimulus selection. However, the functional properties of Imc neurons are not well understood. Here, with electrophysiological experiments in the barn owl Imc, we show that Imc neurons themselves exhibit signatures of stimulus competition. Distant competing stimuli outside the spatial receptive field (RF) suppressed powerfully, and divisively, the responses of Imc neurons to stimuli inside the RF, and did so from all tested locations along the elevation as well as azimuth. Notably, this held true even for locations encoded by the opposite side of the brain from the one containing the recording site. This global divisive inhibition operated independently of the sensory modality of the competing stimulus. Thus, the Imc not only supplies inhibition to the OT to support competition there, but may itself be an active site of stimulus competition. These results from experiments in the barn owl shed light on the functional properties of a vital node in the vertebrate midbrain selection network.

Consequences of dosing and timing on the antibacterial effects of ADEP antibiotics

Antibiotic acyldepsipeptides (ADEPs) exert potent antibacterial activity in rodent models of bacterial infection and exceptional efficacy against persister cells of methicillin-resistant Staphylococcus aureus (MRSA). The mechanism of ADEP action is unusual in that the antibiotic releases the destructive capacity of over-activated ClpP, the proteolytic core of the bacterial Clp protease. The essential bacterial cell division protein FtsZ had emerged in a previous study as a preferred protein substrate of ADEP-activated ClpP but it is definitely not the only cellular substrate.In the current study, we set out to follow the morphological changes that lead to ADEP-mediated bacterial death in S. aureus and Bacillus subtilis, differentiating between antibacterial effects at low and high ADEP concentrations. Here, fluorescence and time-lapse microscopy data show that cells adopt a characteristic phenotype of cell division inhibition at ADEP levels close to the MIC, but retain the capacity to form viable daughter cells for a substantial period of time when transferred to ADEP-free growth medium. After extended exposure to low ADEP concentrations, nucleoids of B. subtilis started to disorganize and upon compound removal many cells failed to re-organize nucleoids, re-initiate cytokinesis and consequently died. Survival versus cell death of filamentous cells attempting recovery depended on the timing of completion of new septa in relation to the loss of cell envelope integrity. We show that the potential to recover after ADEP removal depends on the antibiotic concentration as well as the treatment duration. When exposed to ADEP at concentrations well above the MIC, biomass production ceased rapidly as did the potential to recover. In time-kill studies both long-time exposure to low ADEP levels as well as short-time exposure to high concentrations proved highly effective, while intermittent concentrations and time frames were not. We here provide new insights into the antimicrobial activity of ADEP antibiotics and the consequences of dosing and timing for bacterial physiology which should be considered in view of a potential therapeutic application of ADEPs.

FtsZ-Independent Mechanism of Division Inhibition by the Small Molecule PC190723 in Escherichia coli.

While cell division is a critical process in cellular proliferation, very few antibiotics have been identified that target the bacterial cell-division machinery. Recent studies have shown that the small molecule PC190723 inhibits cell division in several Gram-positive bacteria, with a hypothesized mechanism of action involving direct targeting of the tubulin homolog FtsZ, which is essential for division in virtually all bacterial species. Here, it is shown that PC190723 also inhibits cell division in the Gram-negative bacterium Escherichia coli if the outer membrane permeability barrier is compromised genetically or chemically. The results show that the equivalent FtsZ mutations conferring PC190723 resistance in Staphylococcus aureus do not protect E. coli against PC190723, and that suppressors of PC190723 sensitivity in E. coli, which do not generically decrease outer membrane permeability, do not map to FtsZ or other division proteins. These suppressors display a wide range of morphological and growth phenotypes, and one exhibits a death phenotype in the stationary phase similar to that of a mutant with disrupted lipid homeostasis. Finally, a complementing FtsZ-msfGFP fusion is used to show that PC190723 does not affect the Z-ring structure. Taken together, the findings suggest that PC190723 inhibits growth and division in E. coli without targeting FtsZ. This study highlights the importance of utilizing a combination of genetic, chemical, and single-cell approaches to dissect the mechanisms of action of new antibiotics, which are not necessarily conserved across bacterial species.

Lactate-Dependent Chaperone-Mediated Autophagy Induces Oscillatory Hif-1α Activity Promoting Proliferation of Hypoxic Cells

Response to hypoxia is a highly regulated process controlling multiple cell and tissue level functions. However, we still know little about dynamic single cell responses to hypoxic conditions. Here, using fluorescent reporters of hypoxia response factor-1α (HIF-1α) activity, we show that hypoxic responses in individual cells can be highly dynamic and variable across the population. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, demonstrating that the oscillations of HIF-1α activity and abundance are controlled by the reactive oxygen species-dependent chaperone-mediated autophagy in a respiring subset of cells. Furthermore, we find that the oscillatory response is modulated by the abundance of extracellular lactate in a quorum sensing-like mechanism. We show that oscillatory HIF-1α activity can help rescue hypoxia-mediated inhibition of cell division, suggesting a mechanism for aggressive behavior in a subset of hypoxic tumor cells.

Preserving Mitochondrial Structure and Motility Promotes Recovery of White Matter After Ischemia.

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.

Divisive Inhibition Prevails During Simultaneous Optogenetic Activation of All Interneuron Subtypes in Mouse Primary Visual Cortex

The mouse primary visual cortex (V1) has become an important brain area for exploring how neural circuits process information. Optogenetic tools have helped to outline the connectivity of a local V1 circuit comprising excitatory pyramidal neurons and several genetically-defined inhibitory interneuron subtypes that express parvalbumin, somatostatin, or vasoactive intestinal peptide. Optogenetic modulation of individual interneuron subtypes can alter the visual responsiveness of pyramidal neurons with distinct forms of inhibition and disinhibition. However, different interneuron subtypes have potentially opposing actions, and the potency of their effects relative to each other remains unclear. Therefore, in this study we simultaneously optogenetically activated all interneuron subtypes during visual processing to explore whether any single inhibitory effect would predominate. This aggregate interneuron activation consistently inhibited pyramidal neurons in a divisive manner, which was essentially identical to the pattern of inhibition produced by activating parvalbumin-expressing interneurons alone.

Spatial summation of broadband contrast.

Spatial summation of luminance contrast signals has historically been psychophysically measured with stimuli isolated in spatial frequency (i.e., narrowband). Here, we revisit the study of spatial summation with noise patterns that contain the naturalistic 1/fα distribution of contrast across spatial frequency. We measured amplitude spectrum slope (α) discrimination thresholds and verified if sensitivity to α improved according to stimulus size. Discrimination thresholds did decrease with an increase in stimulus size. These data were modeled with a summation model originally designed for narrowband stimuli (i.e., single detecting channel; Baker & Meese, 2011; Meese & Baker, 2011) that we modified to include summation across multiple-differently tuned-spatial frequency channels. To fit our data, contrast gain control weights had to be inversely related to spatial frequency (1/f); thus low spatial frequencies received significantly more divisive inhibition than higher spatial frequencies, which is a similar finding to previous models of broadband contrast perception (Haun & Essock, 2010; Haun & Peli, 2013). We found summation across spatial frequency channels to occur prior to summation across space, channel summation was near linear and summation across space was nonlinear. Our analysis demonstrates that classical psychophysical models can be adapted to computationally define visual mechanisms under broadband visual input, with the adapted models offering novel insight on the integration of signals across channels and space.

Antibacterial evaluation and mode of action study of BIMQ, a novel bacterial cell division inhibitor

The worldwide spreading of antibiotic resistant bacteria is currently an extremely serious health risk and therefore to develop new antibiotics is an urgent need. In the present study, the antibacterial activity of a new indolyl quinolinium compound and its underline mechanism were investigated. The compound shows an outstanding antibacterial activity against the tested Gram-positive bacteria. The MIC values are in the range of 1–4 μg/mL. The elongation of B. subtilis cells indicates that the compound can inhibit cell division effectively. In addition, the biochemical studies prove that the compound is able to disrupt FtsZ polymerization effectively through a stimulatory mechanism. Furthermore, the compound can delay the development of drug resistance mutants.

Mitochondrial Division Inhibitor 1 Attenuates Mitophagy in a Rat Model of Acute Lung Injury.

The regulation of intracellular mitochondria degradation is mediated by mitophagy. While studies have shown that mitophagy can lead to mitochondrial dysfunction and cell damage, the role of Mdivi-1 and mitophagy remains unclear in acute lung injury (ALI) pathogenesis. In this study, we demonstrated that Mdivi-1, which is widely used as an inhibitor of mitophagy, ameliorated acute lung injury assessed by HE staining, pulmonary microvascular permeability assay, measurement of wet/dry weight (W/D) ratio, and oxygenation index (PaO2/FiO2) analysis. Then, the mitophagy related proteins were evaluated by western blot. The results indicated that LPS-induced activation of mitophagy was inhibited by Mdivi-1 treatment. In addition, we found that Mdivi-1 protected A549 cells against LPS-induced mitochondrial dysfunction. We also found that Mdivi-1 reduced pulmonary cell apoptosis in the LPS-challenged rats and protected pulmonary tissues from oxidative stress (represented by the content of superoxide dismutase, malondialdehyde and lipid peroxides in lung). Moreover, Mdivi-1 treatment ameliorated LPS-induced lung inflammatory response and cells recruitment. These findings indicate that Mdivi-1 mitigates LPS-induced apoptosis, oxidative stress, and inflammation in ALI, which may be associated with mitophagy inhibition. Thus, the inhibition of mitophagy may represent a potential therapy for treating ALI.

Pharmacophore based approach to screen and evaluate novel Mycobacterium cell division inhibitors targeting FtsZ – A modelling and experimental study

Tuberculosis, caused by Mycobacterium tuberculosis has been one of the primal afflictions to human, and owing to the current scenario of drug resistance, newer drugs, and alternate targets are required to mitigate the disease. FtsZ is a GTP hydrolyzing protein, conserved in prokaryotes that plays a central role in Z-ring formation during cell division cytokinesis stage. This study employs the use of pharmacophore models derived from two different datasets based on Mtb-FtsZ GTPase inhibition and whole cell antibacterial activity, to virtually screen and shortlist novel compounds from In-house small molecule library as Mtb-FtsZ inhibitors and evaluate their in-vitro and ex-vivo activity. The results revealed Piperine (IC50 = 21.2 ± 0.7 μM), 4-Bromo di-methoxy coumarin (IC50 = 13.0 ± 1.6 μM) and Di-ethyl amino methyl coumarin (IC50 = 19.4 ± 1.1) as potent Mtb-FtsZ GTPase inhibitors which showed considerable antibacterial activity (84.0 ± 2.6 μM, 56.0 ± 4.3 μM and 108 ± 7.1 μM respectively) against M. smegmatis. They appear to be bacteriostatic, as well as treatment with these compounds led to a 3× increase in cell length of M. smegmatis. Further these molecules also altered the FtsZ gene expression by 3-fold when compared to untreated. In addition compound Aloin, an Aloe exudate showed potent Mtb-FtsZ inhibition (IC50 = 16.7 ± 0.4 μM) but exhibited poor anti-mycobacterial activity (>500 μM).

The toxic mechanisms of BDE-47 to the marine diatom Thalassiosira pseudonana-a study based on multiple physiological processes

Polybrominated diphenyl ethers (PBDEs), a series of highly persistent organic pollutants (POPs), are ubiquitous in marine ecosystems. As key primary producers, microalgae are of great importance on evaluating the environmental outcome of PBDEs pollution. In this study, the toxic mechanisms of BDE-47 on the marine diatom Thalassiosira pseudonana were evaluated by measuring multiple physiological processes. Three concentrations of BDE-47 (25, 15 and 5 μg L−1) were used along with two controls (blank: no BDE-47 or DMSO; negative control: only DMSO). Experiments lasted 144 h (6 days), in which the actual BDE-47 concentrations, cell densities, nutrient (nitrate and phosphate) uptake, pigment compositions, photosynthetic physiology, cell morphology and cellular contents (organic carbon and nitrogen) were measured at 12–48 h intervals. The toxic mechanisms of BDE-47 on T. pseudonana cells were evaluated by measuring multiple physiological processes including photosynthesis, nutrient uptake, cellular material synthesis and cell cycle progressions. The cell divisions of T. pseudonana were severely inhibited by the stress of BDE-47, but the photosynthetic parameters were much less declined and recovered earlier than the cell divisions in the same BDE-47 treatments. The unsuppressed uptake rates of nutrients, increased cell volume and cellular contents indicated the cellular material synthesis proceeded normally. Finally, we found that the cell cycle was arrested in G2/M phase under the stress of BDE-47, we thus concluded that the inhibition of cell divisions by BDE-47 was not due to the lack of energy or cellular materials, where the cell cycle arrest happened; this might be the most important toxicological outcome.

Carbon, Nitrogen and Phosphorous assimilation in V. cholerae – a case of imperfect nutrient sensing

Vibrio cholerae experiences frequent feast and famine conditions. In the absence of all the three nutrient components (Carbon, Nitrogen, and Phosphorous), the cells could manage starvation and survive up to 6 months with only 1 log reduction in viability. Addition of carbon or nitrogen to the starvation media resulted in 3 log reduction in cell number. Simultaneous addition of carbon and nitrogen (Phosphorous starvation) reduced the cell viability to a below detectable level. The suboptimal growth conditions; non-metabolizable source of carbon and nitrogen; variable C: N ratio; inhibition of metabolism and cell division, increased the cell survival under phosphate starvation. However, when all the three components are limited, the cells did not initiate active metabolism and could conserve the energy for long-term survival. This observation suggests that integration of carbon, nitrogen and phosphorus sensing is imperfect in V. cholerae and it cannot down-regulate the metabolism during phosphorous limitation. The carbon and nitrogen could prime the cells to accelerate the rate of metabolism, irrespective of the presence of phosphorous, thereby creating an energetically unfavorable situation. The lack of crucial component phosphorus fails to activate a stringent response that results in increased futile cycling of nutrients, loss of ATP and cell death. The Vibrio genus was found to be less efficient in surviving phosphate starvation than E. coli and S. Typhimurium. The two-component system CreC (a response regulator of phosphorous) is absent in V. cholerae and may be responsible for the lack of stringent response to phosphorus starvation.

Exfoliated Black Phosphorus Promotes in Vitro Bone Regeneration and Suppresses Osteosarcoma Progression through Cancer-Related Inflammation Inhibition.

Nowadays chemotherapy is the main treatment for osteosarcoma disease, even if limited by the lack of selectivity between healthy and cancer cells during the inhibition of cell division. Herein, we propose the use of few-layer two-dimensional black phosphorous (2D bP) as an alternative tool for osteosarcoma treatment and report how 2D bP can stimulate newly forming bone tissue generation after osteosarcoma resection. In our study, we have developed an in vitro model to evaluate the efficacy of 2D bP material with and without near-infrared light irradiation treatment, and we have demonstrated that the presence of 2D bP without treatment inhibits the metabolic activity of osteosarcoma cells (SAOS-2) while inducing both the proliferation and the osteogenic differentiation of human preosteoblast cells (HOb) and mesenchymal stem cells. Furthermore, we also propose an in vitro coculture model (SAOS-2 and HOb cell lines) in order to study the effect of 2D bP on inflammatory response related to cancer. On this coculture model, 2D bP may increase anti-inflammatory cytokine generation (i.e., interleukin-10) and inhibit proinflammatory mediators synthesis (i.e., interleukin-6), thus suggesting the opportunity to prevent cancer-related inflammation. Finally, we have demonstrated that 2D bP represents a promising candidate for future regenerative medicine and anticancer applications.

Nuclear factor (erythroid-derived 2)-like 2 antioxidative response mitigates cytoplasmic radiation-induced DNA double-strand breaks.

It has been reported that DNA double‐strand breaks (DSB) can be induced by cytoplasm irradiation, and that both reactive free radicals and mitochondria are involved in DSB formation. However, the cellular antioxidative responses that are stimulated and the biological consequences of cytoplasmic irradiation remain unknown. Using the Single Particle Irradiation system to Cell (SPICE) proton microbeam facility at the National Institute of Radiological Sciences ([NIRS] Japan), the response of nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) antioxidative signaling to cytoplasmic irradiation was studied in normal human lung fibroblast WI‐38 cells. Cytoplasmic irradiation stimulated the localization of NRF2 to the nucleus and the expression of its target protein, heme oxygenase 1. Activation of NRF2 by tert‐butylhydroquinone mitigated the levels of DSB induced by cytoplasmic irradiation. Mitochondrial fragmentation was also promoted by cytoplasmic irradiation, and treatment with mitochondrial division inhibitor 1 (Mdivi‐1) suppressed cytoplasmic irradiation‐induced NRF2 activation and aggravated DSB formation. Furthermore, p53 contributed to the induction of mitochondrial fragmentation and activation of NRF2, although the expression of p53 was significantly downregulated by cytoplasmic irradiation. Finally, mitochondrial superoxide (MitoSOX) production was enhanced under cytoplasmic irradiation, and use of the MitoSOX scavenger mitoTEMPOL indicated that MitoSOX caused alterations in p53 expression, mitochondrial dynamics, and NRF2 activation. Overall, NRF2 antioxidative response is suggested to play a key role against genomic DNA damage under cytoplasmic irradiation. Additionally, the upstream regulators of NRF2 provide new clues on cytoplasmic irradiation‐induced biological processes and prevention of radiation risks.

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo.

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro-inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)-expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS-mediated Toll-like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi-1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi-1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi-1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi-1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro-inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation.

Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Tubulointerstitial inflammation plays a critical role in the progression of diabetic nephropathy (DN), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes contribute to renal interstitial inflammation in DN. Decreased expression of optineurin (OPTN) is also associated with the progression of DN. We investigated the role of OPTN in activation of the NLRP3 inflammasome in DN. We initially examined the renal biopsy tissues of 172 patients with type 2 DN and 32 nondiabetic patients with renal hamartoma. Expression of renal OPTN was significantly lower in patients with DN and negatively correlated with urinary levels of IL-1β and IL-18. Confocal microscopy analysis of the biopsies indicated no NLRP3 or IL-1β staining in OPTN-positive renal tubular epithelial cells (RTECs), indicating a negative correlation of OPTN expression with the activation of NLRP3 inflammasome. In vitro studies of murine RTECs indicated the levels of OPTN mRNA and protein decreased significantly after stimulation by a high glucose (HG) treatment. Relative to RTECs given HG, RTECs overexpressing OPTN showed significantly lower levels of NLRP3 expression, cleavage of caspase-1 and IL-1β, and release of IL-1 β and IL-18. Overexpression of OPTN in the presence of HG significantly increased the costaining of microtubule-associated protein 1A/1B-light chain 3-II and translocase of outer mitochondrial membrane 20 in RTECs, suggesting that OPTN enhances mitophagy. In addition, mitochondrial division inhibitor 1 blocked the inhibitory effect of OPTN overexpression on the activation of NLRP3 inflammasome in the presence of HG, indicating that OPTN overexpression inhibited NLRP3 inflammasome activation by enhancement of mitophagy. OPTN gene silencing significantly enhanced production of mitochondrial reactive oxygen species (mtROS) in the presence of HG. Compared with HG+OPTN small interfering RNA (siRNA)-treated RTECs, HG+OPTN siRNA+MitoTempo-treated RTECs attenuated NLRP3 inflammasome activation, suggesting that OPTN gene silencing activates the NLRP3 inflammasome by increasing mtROS in HG-treated RTECs. Taken together, our results demonstrate that OPTN inhibits the activation of NLRP3 inflammasome by enhancing mitophagy.-Chen, K., Feng, L., Hu, W., Chen, J., Wang, X., Wang, L., He, Y. Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Theaflavins Improve Insulin Sensitivity through Regulating Mitochondrial Biosynthesis in Palmitic Acid-Induced HepG2 Cells.

Theaflavins, the characteristic and bioactive polyphenols in black tea, possess the potential improving effects on insulin resistance-associated metabolic abnormalities, including obesity and type 2 diabetes mellitus. However, the related molecular mechanisms are still unclear. In this research, we investigated the protective effects of theaflavins against insulin resistance in HepG2 cells induced by palmitic acid. Theaflavins significantly increased glucose uptake of insulin-resistant cells at noncytotoxic doses. This activity was mediated by upregulating the total and membrane bound glucose transporter 4 protein expressions, increasing the phosphor-Akt (Ser473) level, and decreasing the phosphorylation of IRS-1 at Ser307. Moreover, theaflavins were found to enhance the mitochondrial DNA copy number, down-regulate the PGC-1β mRNA level and increase the PRC mRNA expression. Mdivi-1, a selective mitochondrial division inhibitor, could attenuate TFs-induced promotion of glucose uptake in insulin-resistant HepG2 cells. Taken together, these results suggested that theaflavins could improve hepatocellular insulin resistance induced by free fatty acids, at least partly through promoting mitochondrial biogenesis. Theaflavins are promising functional food ingredients and medicines for improving insulin resistance-related disorders.

Specific regions of the SulA protein recognized and degraded by the ATP-dependent ClpYQ (HslUV) protease in Escherichia coli

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ for degradation. However, it is not clear how the natural substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ proteases can degrade SulA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139th∼149th aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142th and 147th residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.