Physical and Functional Interaction of Sequestosome 1 with Keap1 Regulates the Keap1-Nrf2 Cell Defense Pathway

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.     

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.     

Sensing and Responding to Membrane Tension: The Bacterial MscL Channel as a Model System

Mechanosensors are important for many life functions, including the senses of touch, balance, and proprioception; cardiovascular regulation; kidney function; and osmoregulation. Many channels from an assortment of families are now candidates for eukaryotic mechanosensors and proprioception, as well as cardiovascular regulation, kidney function, and osmoregulation. Bacteria also possess two families of mechanosensitive channels, termed MscL and MscS, that function as osmotic emergency release valves. Of the two channels, MscL is the most conserved, most streamlined in structure, and largest in conductance at 3.6 nS with a pore diameter in excess of 30 Å; hence, the structural changes required for gating are exaggerated and perhaps more easily defined. Because of these properties, as well as its tractable nature, MscL represents a excellent model for studying how a channel can sense and respond to biophysical changes of a lipid bilayer. Many of the properties of the MscL channel, such as the sensitivity to amphipaths, a helix that runs along the membrane surface and is connected to the pore via a glycine, a twisting and turning of the transmembrane domains upon gating, and the dynamic changes in membrane interactions, may be common to other candidate mechanosensors. Here we review many of these properties and discuss their structural and functional implications.     

Nitrate Reductase is Needed for Methyl Jasmonate-Mediated Arsenic Toxicity Tolerance of Rice by Modulating the Antioxidant Defense System,Glyoxalase System and Arsenic Sequestration Mechanism

Journal of Plant Growth Regulation - The study was conducted to consider the role of nitrate reductase (NR)-synthesized nitric oxide (NO) in the methyl jasmonate (MJ)-induced tolerance of arsenic...     

Development of an HbA1c-Based Conversion Equation for Estimating Glycated Albumin in a Korean Population with a Wide Range of Glucose Intolerance

Background

Compared to the golden standard glycation index of HbA1c, glycated albumin (GA) has potentials for assessing insulin secretory dysfunction and glycemic fluctuation as well as predicting diabetic vascular complications. However, the reference ranges of GA and a conversion equation need to be clearly defined. We designed this study to determine the reference ranges in patients with normal glucose tolerance (NGT) based on conventional measures of glycemic status and to devise a conversion equation for calculating HbA1c and GA in a Korean population.

Methodology/Principal Findings

In this multicenter, retrospective, cross-sectional study, we recruited antidiabetic drug-naïve patients with available glycemic variables including HbA1c, GA, and fasting plasma glucose regardless of glucose status. For the reference interval of serum GA, 5^th to 95^th percentile value of GA in subjects with NGT was adopted. The conversion equation between HbA1c and GA was devised using an estimating regression model with unknown break-points method. The reference range for GA was 9.0–14.0% in 2043 subjects. The 95^th percentile responding values for FPG, and HbA1c were approximately 5.49 mmol/l, and 5.6%, respectively. The significant glycemic turning points were 5.868% HbA1c and 12.2% GA. The proposed conversion equation for below and above the turning point were GA (%) = 6.960+0.8963 × HbA1c (%) and GA (%) = −9.609+3.720 × HbA1c (%), respectively.

Conclusions/Significance

These results should be helpful in future studies on the clinical implications of high GA relative to HbA1c and the clinical implementation of diabetes management.     

The Ets Transcription Factor GABP Is a Component of the Hippo Pathway Essential for Growth and Antioxidant Defense

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The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein''s N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.     

Erratum to: Quantum Chemical Calculations on Two Compounds of Proquazone and Proquazone Type Calcites as a Calcium Sensing Receptor (CaSR) Inhibitory Profiles

Russian Journal of Bioorganic Chemistry - An Erratum to this paper has been published: https://doi.org/10.1134/S106816202134001X     

A Chemical Genetic Screen for Modulators of Exocytic Transport Identifies Inhibitors of a Transport Mechanism Linked to GTR2 Function

Membrane and protein traffic to the cell surface is mediated by partially redundant pathways that are difficult to perturb in ways that yield a strong phenotype. Such robustness is expected in a fine-tuned process, regulated by environmental cues, that is required for controlled cell surface growth and cell proliferation. Synthetic genetic interaction screens are especially valuable for investigating complex processes involving partially redundant pathways or mechanisms. In a previous study, we used a triple-synthetic-lethal yeast mutant screen to identify a novel component of the late exocytic transport machinery, Avl9. In a chemical-genetic version of the successful mutant screen, we have now identified small molecules that cause a rapid (within 15 min) accumulation of secretory cargo and abnormal Golgi compartment-like membranes at low concentration (<2 μM), indicating that the compounds likely target the exocytic transport machinery at the Golgi. We screened for genes that, when overexpressed, suppress the drug effects, and found that the Ras-like small GTPase, Gtr2, but not its homolog and binding partner, Gtr1, efficiently suppresses the toxic effects of the compounds. Furthermore, assays for suppression of the secretory defect caused by the compounds suggest that Gtr proteins can regulate a pathway that is perturbed by the compounds. Because avl9Δ and gtr mutants share some phenotypes, our results indicate that the small molecules identified by our chemical-genetic strategy are promising tools for understanding Avl9 function and the mechanisms that control late exocytic transport.Cell growth and proliferation, as well as the regulation of cell surface composition, are achieved by an intracellular transport machinery that delivers proteins and membrane to the cell surface. The transport machinery is regulated by environmental sensing and signaling pathways that are integrated for the fine-tuned control of transport to the cell surface. The mechanisms that regulate cell growth and proliferation are highly robust; therefore, they can function in a wide range of environmental conditions and even when some components of the transport or signaling machinery fail. In eukaryotic cells, this robustness is achieved in part by a complex network of membrane and protein traffic routes to the cell surface (17, 33). Defects in a transport pathway can result in cargo transport by an alternate route, making transport defects difficult to detect in mutant screens (17, 18). Therefore, relatively little is known about the mechanisms by which protein and membrane cargo is transported from late exocytic sorting compartments, the late Golgi compartments and endosomes, and we have yet to identify most of the components that mediate and regulate this process.Complex processes are more readily understood in relatively simple organisms. For this reason, the budding yeast Saccharomyces cerevisiae has become one of the most powerful experimental models for understanding intracellular transport, and most of the conserved components of the exocytic traffic machinery were first discovered by using yeast genetic strategies (27). We used a yeast genetic screen to identify a novel component of the late exocytic transport machinery, Avl9, a member of an ancient eukaryotic protein superfamily (18). Avl9 is essential in a mutant strain lacking Vps1, a dynamin homolog that is thought to function in transport vesicle formation at a late Golgi compartment (26, 34), and also lacking Apl2, a large subunit of the adaptor protein 1 (AP-1) complex, which is required for forming certain classes of clathrin-coated vesicles at late Golgi compartments and endosomes (18, 19, 31, 42). The apl2Δ and vps1Δ mutants have defects in an exocytic pathway(s), but these mutants, as well as an apl2Δ vps1Δ double mutant, grow well because cargo is rerouted into a remaining pathway(s) (18). Mutations such as avl9Δ, which are lethal in an apl2Δ vps1Δ strain but not in a wild-type strain, are expected to cause defects in a branch of the exocytic pathway that remains functional in the apl2Δ vps1Δ strain. Analogous to using mutagenesis to screen for a secretory block in the apl2Δ vps1Δ mutant, we performed a high-throughput screen of a large library of small molecules to identify compounds that inhibit the growth of the vps1Δ apl2Δ mutant but which have relatively little effect on wild-type cells. The targets of these compounds are potential components of the secretory machinery, and some of the compounds may interfere with an Avl9-related function. The biochemical function of Avl9 and related proteins is still unknown, and the inhibitors identified by our screen strategy could be valuable tools in understanding the role of Avl9 in both yeast and mammalian cells.Our high-throughput screen was successful in identifying novel exocytic transport inhibitors, and we describe the phenotypic effects of one structurally similar group of compounds in detail. Furthermore, we show that the toxic effects of this group of compounds are inhibited by highly expressing GTR2, which encodes a Ras-like small GTPase that plays a role in regulating nutrient-responsive TORC1 (target of rapamycin complex 1) kinase signaling, exocytic cargo sorting at endosomes, and epigenetic control of gene expression (7, 11, 14, 25, 37). Therefore, the small molecules identified by our chemical-genetic approach are promising tools for understanding how signaling pathways that respond to environmental conditions regulate the traffic pathways that mediate cell growth and proliferation.