Piezo1 acts upstream of TRPV4 to induce pathological changes in endothelial cells due to shear stress [Signal Transduction]

December 9th, 2020 by Sandip M Swain, Rodger A Liddle

The ion channels Piezo1 and TRPV4 have both, independently, been implicated in high venous pressure- and fluid shear stress-induced vascular hyperpermeability in endothelial cells. However, the mechanism by which Piezo1 and TRPV4 channels execute the same function is poorly understood. Here we demonstrate that Piezo1 regulates TRPV4 channel activation in endothelial cells and that Piezo1-mediated TRPV4 channel opening is a function of the strength and duration of fluid shear stress. We first confirmed that either fluid shear stress or the Piezo1 agonist, Yoda1, led to an elevation in intracellular calcium ([Ca2+]i), and that application of the Piezo1 antagonist, GsMTx4, completely blocked this change. We discovered that high and prolonged shear stress caused sustained [Ca2+]i elevation which was blocked by inhibition of TRPV4 channel opening. Moreover, Piezo1 stimulated TRPV4 opening through activation of phospholipase A2. TRPV4-dependent sustained [Ca2+]i elevation was responsible for fluid shear stress- and Piezo1-mediated disruption of adherens junctions and actin remodeling. Blockade of TRPV4 channels with the selective TRPV4 blocker, HC067047, prevented the loss of endothelial cell integrity and actin disruption induced by Yoda1 or shear stress and prevented Piezo1-induced monocyte adhesion to endothelial cell monolayers. These findings demonstrate that Piezo1 activation by fluid shear stress initiates a calcium signal that causes TRPV4 opening which in turn is responsible for the sustained phase calcium elevation that triggers pathological events in endothelial cells. Thus, deleterious effects of shear stress are initiated by Piezo1 but require TRPV4.

Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide [Molecular Biophysics]

October 30th, 2020 by Sungjin Lee, Jeongbeen Heo, Chin-Ju Park

Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. However, the structural details and determinants of the phosphorylated RPA32 – RPA70 interaction are still unknown. In this study, we provide molecular details of the interaction between RPA70 and a mimic of phosphorylated RPA32 (pmRPA32) using fluorescence polarization and NMR analysis. We show that the N-terminal domain of RPA70 (RPA70N) specifically participates in pmRPA32 binding, while the unphosphorylated RPA32 does not bind to RPA70N. Our NMR data revealed that RPA70N binds pmRPA32 using a basic cleft region. We also show that at least 6 negatively charged residues of pmRPA32 are required for RPA70N binding. By introducing alanine mutations into hydrophobic positions of pmRPA32, we found potential points of contact between RPA70N and the N-terminal half of pmRPA32. We used this information to guide docking simulations that suggest the orientation of pmRPA32 in complex with RPA70N. Our study demonstrates detailed features of the domain-domain interaction between RPA70 and RPA32 upon phosphorylation. This result provides insight into how phosphorylation tunes transient bindings between RPA and its partners in DNA resection.

Glycans function as a Golgi export signal to promote the constitutive exocytic trafficking [Membrane Biology]

August 21st, 2020 by Xiuping Sun, Hieng Chiong Tie, Bing Chen, Lei Lu

Most proteins in the secretory pathway are glycosylated. However, the role of glycans in membrane trafficking is still unclear. Here, we discovered that transmembrane secretory cargos, such as interleukin 2 receptor α subunit or Tac, transferrin receptor and cluster of differentiation 8a, unexpectedly displayed substantial Golgi localization when their O-glycosylation was compromised. By quantitatively measuring their Golgi residence times, we found that the observed Golgi localization of O-glycan deficient cargos is due to their slow Golgi export. Using a super-resolution microscopy method that we previously developed, we revealed that O-glycan deficient Tac chimeras localize at the interior of the trans-Golgi cisternae. O-glycans were observed to be both necessary and sufficient for the efficient Golgi export of Tac chimeras. By sequentially introducing O-glycosylation sites to ST6GAL1, we demonstrated that O-glycan’s effect on Golgi export is probably additive. Finally, the finding that N-glycosylated GFP substantially reduces the Golgi residence time of a Tac chimera suggests that N-glycans might have a similar effect. Therefore, both O- and N-glycans might function as a generic Golgi export signal at the trans-Golgi to promote the constitutive exocytic trafficking.

Catastrophic actin filament bursting by cofilin, Aip1, and coronin [Cell Biology]

July 28th, 2020 by Vivian W Tang, Ambika V. Nadkarni, William M Brieher

Cofilin is an actin filament severing protein necessary for fast actin turnover dynamics. Coronin and Aip1 promote cofilin mediated actin filament disassembly, but the mechanism is somewhat controversial. An early model proposed that the combination of cofilin, coronin, and Aip1 disassembled filaments in bursts. A subsequent study only reported severing. Here, we used electron microscopy to show that actin filaments convert directly into globular material. A monomer trap assay also shows that the combination of all three factors produces actin monomers faster than any two factors alone. We show that coronin accelerates the release of inorganic phosphate from actin filaments and promotes highly cooperative cofilin binding to actin to create long stretches of polymer with a hypertwisted morphology. Aip1 attacks these hypertwisted regions along their sides, disintegrating them into monomers or short oligomers. The results are consistent with a catastrophic mode of disassembly, not enhanced severing alone.

Survivin in breast cancer-derived exosomes activates fibroblasts by upregulating SOD1, whose feedback promotes cancer proliferation and metastasis [Molecular Bases of Disease]

July 24th, 2020 by Kangdi Li, Ting Liu, Jie Chen, Huying Ni, Wenhua Li

Cancer-associated fibroblasts (CAFs) play a critical role in the coevolution of breast tumor cells and their microenvironment by modifying cellular compartments and regulating cancer cell functions via stromal-epithelial dialogue. However, the relationship and interaction between stromal and epithelial cells are still poorly understood. Herein, we revealed that breast cancer cells have a stronger ability to activate fibroblasts and transform them into myofibroblasts (CAF-like) than normal breast epithelial cells, and this stronger ability occurs through paracrine signaling. In turn, myofibroblasts promote the proliferation, epithelial-to-mesenchymal transition (EMT), and stemness of breast cancer cells. Detailed regulatory mechanisms showed that, compared to normal cells, Survivin is overexpressed in breast cancer cells and secreted extracellularly in the form of exosomes, which are then internalized by fibroblasts. Breast cancer cell-derived Survivin upregulates SOD1 expression in fibroblasts and then converts them into myofibroblasts, conversely inducing breast cancer progression in vitro and in vivo. Thus, our results indicate that Survivin acts as an activator of the tumor microenvironment and that SOD1 upregulation in fibroblasts can promote breast cancer progression. These results suggest that targeting Survivin and SOD1 may be a potential therapeutic strategy for breast cancer.
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Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F [Molecular Biophysics]

July 24th, 2020 by Trieu Le, Son C Le, Yang Zhang, Pengfei Liang, Huanghe Yang

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid trans-membrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to Annexin V (AnV), a phosphatidylserine (PS) binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.

H2S and reactive sulfur signaling at the host bacterial pathogen interface [Gene Regulation]

July 22nd, 2020 by Brenna J.C. Walsh, David P. Giedroc

Bacterial pathogens that cause invasive disease in the vertebrate host must adapt to host efforts to cripple their viability. Major host insults are reactive oxygen and reactive nitrogen species as well as cellular stress induced by antibiotics. Hydrogen sulfide (H2S) is emerging as an important player in cytoprotection against these stressors, which may well be attributed to downstream more oxidized sulfur species termed reactive sulfur species (RSS). In this review, we summarize recent work that suggests that H2S/RSS impacts bacterial survival in infected cells and animals. We discuss the mechanisms of biogenesis and clearance of RSS in the context of a bacterial H2S/RSS homeostasis model, and the bacterial transcriptional regulatory proteins that act as “sensors” of cellular RSS that maintain H2S/RSS homeostasis. In addition, we cover fluorescence imaging- and mass spectrometry-based approaches used to detect and quantify RSS in bacterial cells. Lastly, we discuss proteome persulfidation (S-sulfuration) as potential mediators of H2S/RSS signaling in bacteria in the context of the writer-reader-eraser paradigm, and progress toward ascribing regulatory significance to this widespread post-translational modification.

Ubiquitination of interleukin-1{alpha} is associated with increased pro-inflammatory polarization of murine macrophages deficient in the E3 ligase ITCH [Cell Biology]

June 25th, 2020 by Xi Lin, Hengwei Zhang, Brendan F. Boyce, Lianping Xing

Macrophages play critical roles in homeostasis and inflammation. Macrophage polarization to either a pro-inflammatory or anti-inflammatory status is controlled by activating inflammatory signaling pathways. Ubiquitination is a post-translational modification that regulates these inflammatory signaling pathways. However, the influence of protein ubiquitination on macrophage polarization has not been well studied. We hypothesized that the ubiquitination status of key proteins in inflammatory pathways contributes to macrophage polarization, which is regulated by itchy E3 ubiquitin ligase (ITCH), a negative regulator of inflammation. Using ubiquitin proteomics, we found that ubiquitination profiles are different among polarized murine macrophage subsets. Interestingly, interleukin-1α (IL-1α), an important proinflammatory mediator, was specifically ubiquitinated in lipopolysaccharide-induced proinflammatory macrophages, which was enhanced in ITCH-deficient macrophages. The ITCH-deficient macrophages had increased levels of the mature form of IL-1α and exhibited proinflammatory polarization, and reduced deubiquitination of IL-1α protein. Finally, IL-1α neutralization attenuated pro-inflammatory polarization of the ITCH-deficient macrophages. In conclusion, ubiquitination of IL-1α is associated with increased pro-inflammatory polarization of macrophages deficient in the E3 ligase ITCH.
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Viperin: an ancient radical-SAM enzyme finds its place in modern cellular metabolism and innate immunity [Immunology]

June 16th, 2020 by Soumi Ghosh, E. Neil G. Marsh

Viperin plays an important and multifaceted role in the innate immune response to viral infection. Viperin is also notable as one of very few radical SAM-dependent enzymes present in higher animals; however, the enzyme appears broadly conserved across all kingdoms of life, which suggests that it represents an ancient defense mechanism against viral infections. Although viperin was discovered some 20 years ago, only recently was the enzyme’s structure determined and its catalytic activity elucidated. The enzyme converts CTP to 3’,4’-didehydro-3’-deoxy-CTP, which functions as novel chain-terminating antiviral nucleotide when misincorporated by viral RNA-dependent RNA polymerases. Moreover, in higher animals, viperin interacts with numerous other host and viral proteins, and it is apparent that this complex network of interactions constitutes another important aspect of the protein’s antiviral activity. An emerging theme is that viperin appears to facilitate ubiquitin-dependent proteasomal degradation of some of the proteins it interacts with. Viperin- targeted protein degradation contributes to the antiviral response either by down-regulating various metabolic pathways important for viral replication or by directly targeting viral proteins for degradation. Here, we review recent advances in our understanding of the structure and catalytic activity of viperin, together with studies investigating the interactions between viperin and its target proteins. These studies have provided detailed insights into the biochemical processes underpinning this unusual enzyme’s wide-ranging antiviral activity. We also highlight recent intriguing reports that implicate a broader role for viperin in regulating non-pathological cellular processes, including thermogenesis and protein secretion.

Roles of active site residues in catalysis, substrate binding, cooperativity and the reaction mechanism of the quinoprotein glycine oxidase [Enzymology]

March 31st, 2020 by Kyle J. Mamounis, Erik T Yukl, Victor L. Davidson

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetremeric enzyme exhibits strong cooperativity towards glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766 and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.
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