The inhibitory role of benzo-dioxole-piperamide on the phosphorylation process as an NF-Kappa B silencer.

NF-κB contributes to the biosynthesis of various chemokines, cytokines, and enzymes. It plays many crucial roles in the upstream neuroinflammatory pathways. Briefly, the inhibitory IkB subunit is cleaved and phosphorylated by the IKK-α/ß enzyme. It leads to the activation and translocation of the NF-κB (p50/p65) complex into the nucleus. Subsequently, the activated NF-κB interacts with the genomic DNA and contributes to expressing various proinflammatory cytokines. In the present study, we developed a novel NF-κB inhibitor encoded (D5) and investigated the efficacy of our druggable compound through several in silico, in vitro, and in situ analysis. The results demonstrated that D5 not only inhibited the mRNA expression of the IKK-α/ß enzyme (around 86-96% suppression rate for both cell lines at 12 and 24 h time frames) but also by interacting to the active site of the mentioned kinase (dock score -6.14 and binding energy -23.60 kcal/mol) reduced the level of phosphorylated IkB-α in the cytosol around 96-99% and p65 subunit in the nucleus around 73-90% (among all groups in 12 and 24 h time points). Additionally, the results indicated that D5 suppressed the NF-κB target mRNA levels of TNF-α and IL-6 in a total average of around 92%. Overall, The results demonstrated that D5 in a considerably lower concentration than Dis (0.71 µM vs. 52.73 µM) showed significantly higher inhibitory efficacy on NF-κB translocation approx. 200-300%. The results suggested D5 as a potent NF-κB silencer, but further investigations are required to validate our outcomes.

Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Mechanisms of Disulfide Bond Formation in Nascent Polypeptides Entering the Secretory Pathway.

Disulphide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulphide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulphide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulphide bond formation is reviewed with emphasis on events that occur during ER entry. Disulphide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulphide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulphide bond formation and identifies key questions that need to be addressed to further understand this fundamental process.

A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

Towards the application of Tc toxins as a universal protein translocation system.

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.

Role of BGS13 in the Secretory Mechanism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels.

The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion.

Mortality and Cancer Incidence in Carriers of Balanced Robertsonian Translocations: A National Cohort Study.

A balanced robertsonian translocation (rob) results from fusion of 2 acrocentric chromosomes. Carriers are phenotypically normal and are often diagnosed because of recurrent miscarriages, infertility, or aneuploid offspring. Mortality and site-specific cancer risks in carriers have not been prospectively investigated. We followed 1,987 carriers diagnosed in Great Britain for deaths and cancer risk, over an average of 24.1 years. Standardized mortality and incidence ratios were calculated comparing the number of observed events against population rates. Overall mortality was higher for carriers diagnosed before age 15 years (standardized mortality ratio (SMR) = 2.00, 95% confidence interval (CI): 1.09, 3.35), similar for those diagnosed aged 15-44 years (SMR = 1.06, 95% CI: 0.86-1.28), and lower for those diagnosed aged 45-84 years (SMR = 0.81, 95% CI: 0.68, 0.95). Cancer incidence was higher for non-Hodgkin lymphoma (standardized incidence ratio (SIR) = 1.90, 95% CI: 1.01, 3.24) and childhood leukemia (SIR = 14.5, 95% CI: 1.75, 52.2), the latter particularly in rob(15;21) carriers (SIR = 447.8, 95% CI: 11.3, 2,495). Rob(13;14) carriers had a higher breast cancer risk (SIR = 1.58, 95% CI: 1.12, 2.15). Mortality risks relative to the population in diagnosed carriers depend on age at cytogenetic diagnosis, possibly reflecting age-specific cytogenetic referral reasons. Carriers might be at greater risk of childhood leukemia and non-Hodgkin lymphoma and those diagnosed with rob(13;14) of breast cancer.

Biosynthesis, structure, and folding of the insulin precursor protein.

Insulin synthesis in pancreatic ß-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic ß-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.

The murine lung as a factory to produce secreted intrapulmonary and circulatory proteins.

We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 µg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.

Involvement of GSK3/ß-catenin in the action of extracellular ATP on differentiation of primary cultures from rat calvaria into osteoblasts.

Modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the involvement of the GSK3/ßcatenin signaling in the action of ATPγ-S on osteogenic differentiation of primary cell cultures from rat calvaria. Our results indicate that the cell treatment with 10 or 100 µM ATPγ-S for 96 h increase the cytoplasmic levels of ß-catenin and its translocation to nucleus respect to control. A similar effect was observed after cell treatment with the GSK3 inhibitor LiCl (10 mM). Cell treatments with 4-10 mM LiCl significantly stimulated ALP activity respect to control at 4 and 7 days, suggesting that inhibition of GSK-3 mediates osteoblastic differentiation of rat calvarial cells. Effects comparison between ATP and LiCl shown that ALP activity was significantly increased by 10 µM ATPγ-S and decreased by 10 mM LiCl at 10 day of treatment, respect to control, suggesting that the effect of ATPγ-S was less potent but more persistent than of LiCl in stimulating this osteogenic marker in calvarial cells. Cell culture mineralization was significantly increased by treatment with 10 µM ATPγ-S and decreased by 10 mM LiCl, respect to control. In together, these results suggest that GSK3 inhibition is involved in ATPγ-S action on rat calvarial cell differentiation into osteoblasts at early steadies. In addition such inhibition by LiCl appear promote osteoblasts differentiation at beginning but has a deleterious effect on its function at later steadies as the extracellular matrix mineralization.

Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

The way is the goal: how SecA transports proteins across the cytoplasmic membrane in bacteria.

In bacteria, translocation of most soluble secreted proteins (and outer membrane proteins in Gram-negative bacteria) across the cytoplasmic membrane by the Sec machinery is mediated by the essential ATPase SecA. At its core, this machinery consists of SecA and the integral membrane proteins SecYEG, which form a protein conducting channel in the membrane. Proteins are recognised by the Sec machinery by virtue of an internally encoded targeting signal, which usually takes the form of an N-terminal signal sequence. In addition, substrate proteins must be maintained in an unfolded conformation in the cytoplasm, prior to translocation, in order to be competent for translocation through SecYEG. Recognition of substrate proteins occurs via SecA-either through direct recognition by SecA or through secondary recognition by a molecular chaperone that delivers proteins to SecA. Substrate proteins are then screened for the presence of a functional signal sequence by SecYEG. Proteins with functional signal sequences are translocated across the membrane in an ATP-dependent fashion. The current research investigating each of these steps is reviewed here.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Phosphorylation of P27 by AKT is required for inhibition of cell cycle progression in cholangiocarcinoma.

BACKGROUND AND OBJECTIVE: P27 is a putative tumor suppressor when located in the nucleus and AKT is an inhibitor of P27 which promotes growth of cholangiocarcinoma. We hypothesized that AKT-dependent phosphorylation at the P27 nuclear localization sequence T157 leads to nuclear export of P27, and thus loss of its tumor suppressive function. This study investigated whether loss of cell cycle regulation in cholangiocarcinoma due to subcellular localization of P27. METHODS: Human cholangiocarcinoma cells were transfected with AKT. P27 was tagged with yellow fluorescence protein. Cell cycle progression was determined by flow cytometry. Migration and invasion of was measured by transwell assay. RESULTS: Overexpression of wildtype P27 or P27-T157A in Mz-ChA-1 cells resulted in G1 arrest; expression of myr-AKT caused translocation of P27-YFP and endogenous P27 from the nucleus to the cytoplasm, leading to inhibition of P27-dependent G1 arrest; the AKT inhibitor and expression of dnAKT increased P27-YFP accumulation in the nucleus and promoted G1 arrest. In contrast, cells expressing YFP-P27-T157A or P27-YFP accumulated only in the nucleus. Co-expression of myr-AKT failed to induce P27-YFP translocation to the cytoplasm or inhibit G1 arrest. Overexpression of P27-T157A significantly increased migration and invasion. CONCLUSIONS: Cholangiocarcinoma growth is associated with nuclear export of P27 that is due to AKT-mediated phosphorylation of P27 at T157.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.

Signal Peptide Hydrophobicity Modulates Interaction with the Twin-Arginine Translocase.

The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in cis to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway.IMPORTANCE Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates.

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum-associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

The cytoplasmic translocation of Cx32 mediates cisplatin resistance in ovarian cancer cells.

Ovarian cancer is the most lethal gynecologic malignancy, and cisplatin is one of the first-line chemotherapeutic agents. However, acquired cisplatin resistance prevents the successful treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. In this study, we established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP. Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in chemoresistance.