Molecular interplay of an assembly machinery for nitrous oxide reductase.

Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased considerably over the last decades1-3. As the final step of bacterial denitrification, N2O is reduced to chemically inert N2 (refs. 1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N2OR) (ref. 5). The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. 4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu+ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N2OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N2OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.

Aurora-A kinase is differentially expressed in the nucleus and cytoplasm in normal Müllerian epithelium and benign, borderline and malignant serous ovarian neoplasms.

BACKGROUND: Aurora-A kinase is important for cellular proliferation and is implicated in the tumorigenesis of several malignancies, including of the ovary. Information regarding the expression patterns of Aurora-A in normal Müllerian epithelium as well as benign, borderline and malignant epithelial ovarian neoplasms is limited. METHODS: We investigated Aurora-A expression by immunohistochemistry in 15 benign, 19 borderline and 17 malignant ovarian serous tumors, and 16 benign, 8 borderline, and 2 malignant ovarian mucinous tumors. Twelve fimbriae from seven patients served as normal Müllerian epithelium controls. We also examined Aurora-A protein expression by western blot in normal fimbriae and tumor specimens. RESULTS: All normal fimbriae (n = 12) showed nuclear but not cytoplasmic Aurora-A immunoreactivity by immunohistochemistry. Benign ovarian tumors also showed strong nuclear Aurora-A immunoreactivity. Forty-eight percent (13/27) of borderline tumors demonstrated nuclear Aurora-A immunoreactivity, while the remainder (52%, 14/27) lacked Aurora-A staining. Nuclear Aurora-A immunoreactivity was absent in all malignant serous tumors, however, 47% (8/17) demonstrated perinuclear cytoplasmic staining. These results were statistically significant when tumor class (benign/borderline/malignant) was compared to immunoreactivity localization or intensity (Fisher Exact Test, p < 0.01). Western blot analysis confirmed the greater nuclear Aurora-A expression in control Müllerian epithelium compared to borderline and malignant tumors. CONCLUSION: Aurora-A kinase is differentially expressed across normal Müllerian epithelium, benign and borderline serous and mucinous ovarian epithelial neoplasms and malignant serous ovarian tumors., with nuclear expression of unphosphorylated Aurora-A being present in normal and benign neoplastic epithelium, and lost in malignant serous neoplasms. Further studies of the possible biological and clinical implications of the loss of nuclear Aurora-A expression in ovarian tumors, and its role in ovarian carcinogenesis are warranted.

Anaplastic Lymphoma Kinase Overexpression Is Associated with Aggressive Phenotypic Characteristics of Ovarian High-Grade Serous Carcinoma.

Deregulated full-length anaplastic lymphoma kinase (ALK) overexpression has been found in some primary solid tumors, but little is known about its role in ovarian high-grade serous carcinoma (HGSC). The current study focused on the functional roles of ALK in HGSC. Cytoplasmic ALK immunoreactivity without chromosomal rearrangement and gene mutations was significantly higher in HGSC compared with non-HGSC-type ovarian carcinomas, and was significantly associated with several unfavorable clinicopathologic factors and poor prognosis. HGSC cell lines stably overexpressing ALK exhibited increased cell proliferation, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Expression of the nervous system-associated gene, ELAVL3, and the corresponding protein (commonly known as HuC) was significantly increased in cells overexpressing ALK. Expression of SRY-box transcription factor (Sox)2 and Sox3 (genes associated with the neural progenitor population) increased in ALK-overexpressing but not ALK-knockdown cells. Furthermore, overexpression of Sox2 or Sox3 enhanced both ALK and ELAVL3 promoter activities, suggesting the existence of ALK/Sox/HuC signaling loops. Finally, ALK overexpression was attributed to increased expression of neuroendocrine markers, including synaptophysin, CD56, and B-cell lymphoma 2, in HGSC tissues. These findings suggest that overexpression of full-length ALK may influence the biological behavior of HGSC through cooperation with ELAVL3 and Sox factors, leading to the establishment and maintenance of the aggressive phenotypic characteristics of HGSC.

FGGY carbohydrate kinase domain containing is expressed and alternatively spliced in skeletal muscle and attenuates MAP kinase and Akt signaling.

Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.

Nuclear Transglutaminase 2 interacts with topoisomerase II⍺ to promote DNA damage repair in lung cancer cells.

BACKGROUND: To block repairs of DNA damages, especially the DNA double strand break (DSB) repair, can be used to induce cancer cell death. DSB repair depends on a sequential activation of DNA repair factors that may be potentially targeted for clinical cancer therapy. Up to now, many protein components of DSB repair complex remain unclear or poorly characterized. In this study, we discovered that Transglutaminase 2 (TG2) acted as a new component of DSB repair complex. METHODS: A bioinformatic analysis was performed to identify DNA damage relative genes from dataset from The Cancer Genome Atlas. Immunofluorescence and confocal microscopy were used to monitor the protein localization and recruitment kinetics. Furthermore, immunoprecipitation and mass spectrometry analysis were performed to determine protein interaction of both full-length and fragments or mutants in distinct domain. In situ lung cancer model was used to study the effects cancer therapy in vivo. RESULTS: After DSB induction, cytoplasmic TG2 was extensively mobilized and translocated into nucleus after phosphorylated at T162 site by DNA-PKcs. Nuclear TG2 quickly accumulated at DSB sites and directly interacting with Topoisomerase IIα (TOPOIIα) with its TGase domain to promote DSB repair. TG2 deficient cells lost capacity of DSB repair and become susceptible to ionizing radiation. Specific inhibition of TG2-TOPOIIα interaction by glucosamine also significantly inhibited DSB repair, which increased sensitivity in lung cancer cells and engrafted lung cancers. CONCLUSIONS: These findings elucidate new mechanism of TG2 in DSB repair trough directly interacting with TOPOIIα, inhibition of which provided potential target for overcoming cancer resistance.

OsUEV1B, an Ubc enzyme variant protein, is required for phosphate homeostasis in rice.

Phosphorus is a crucial macronutrient for plant growth and development. The mechanisms for maintaining inorganic phosphate (Pi) homeostasis in rice are not well understood. The ubiquitin-conjugating enzyme variant protein OsUEV1B was previously found to interact with OsUbc13 and mediate lysine63-linked polyubiquitination. In the present study, we found OsUEV1B was specifically inhibited by Pi deficiency, and was localized in the nucleus and cytoplasm. Both osuev1b mutant and OsUEV1B-RNA interference (RNAi) lines displayed serious symptoms of toxicity due to Pi overaccumulation. Some Pi starvation inducible and phosphate transporter genes were upregulated in osuev1b mutant and OsUEV1B-RNAi plants in association with enhanced Pi acquisition, and representative Pi starvation responses, including stimulation of acid phosphatase activity and root hair growth, were also activated in the presence of sufficient Pi. A yeast two-hybrid screen revealed an interaction between OsUEV1B and OsVDAC1, which was confirmed by bimolecular fluorescence complementation and firefly split-luciferase complementation assays. OsVDAC1 encoded a voltage-dependent anion channel protein localized in the mitochondria, and OsUbc13 was shown to interact with OsVDAC1 via yeast two-hybrid and bimolecular fluorescence complementation assays. Under sufficient Pi conditions, similar to osuev1b, a mutation in OsVDAC1 resulted in significantly greater Pi concentrations in the roots and second leaves, improved acid phosphatase activity, and enhanced expression of the Pi starvation inducible and phosphate transporter genes compared with wild-type DongJin, whereas overexpression of OsVDAC1 had the opposite effects. OsUEV1B or OsVDAC1 knockout reduced the mitochondrial membrane potential and adenosine triphosphate levels. Moreover, overexpression of OsVDAC1 in osuev1b partially restored its high Pi concentration to a level between those of osuev1b and DongJin. Our results indicate that OsUEV1B is required for rice phosphate homeostasis.

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases.

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

Determining the prognostic significance of IKKα in prostate cancer.

BACKGROUND: As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome. METHODS: A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. RESULTS: High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. CONCLUSION: The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.

Post-translational Modifications of Fumarase Regulate its Enzyme Activity and Function in Respiration and the DNA Damage Response.

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.

CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides.

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2 These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.

Attenuation of Rat Colon Carcinogenesis by Styela plicata Aqueous Extract. Modulation of NF-κB Pathway and Cytoplasmic Sod1 Gene Expression.

OBJECTIVE: In search for a unique natural combination of highly active biological components for treatment against colon cancer, we used aqueous extract of Ascidia, Styela plicata (ASCex), a marine invertebrate depending on its richness of high levels of biologically active components as indicated in our previous studies, against rat colon cancer, exploring its underlying mechanisms. METHODS: Rats chemically initiated for colon cancer were either non-treated or post-treated with highly saturated ASCex for 32 weeks after initiation, other groups of rats were administered ASCex without cancer initiation or served as normal controls. RESULTS: Rats treated with ASCex alone did not show any signs of non-favored health conditions. Treatment with ASCex after cancer initiation has significantly reduced the average incidences, multiplicities and volumes of colon tumors (adenomas and adenocarcinomas) as compared with the non-treated cancer group. ASCex has also significantly reduced the total numbers of aberrant crypt foci (ACF), surrogate biomarkers for colon cancer as compared with the non-treated cancer group. Moreover, anti-proliferative celluar nucular antigen (PCNA) immunohistochemical staining revealed that ASCex exerted significant antiproliferative characteristics in the carcinogen-treated colonic mucosa as compared with its corresponding control. Also, treatment with ASCex has markedly down-regulated the mRNA expression levels of Nuclear Factor-kappa B (NF-κB), a nuclear transcriptional activator as well as the mRNA expression of the cytoplasmic SOD1 gene which encodes Cu/Zn SOD, the first line defense against superoxide radicals. CONCLUSION: Collectively, ASCex could act as a potent chemotherapeutic drug against colon cancer, likely through the influence of its rich active metabolites which interfere with various biological pathways including inhibition of protein synthesis during cellular growth and marked induction of antioxidative capacity in the colonic mucosa. This role has been extensively discussed herein.

Peroxiredoxin 1 plays a primary role in protecting pancreatic ß-cells from hydrogen peroxide and peroxynitrite.

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

Spatially regulated editing of genetic information within a neuron.

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.

Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines.

Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. In comparison, little is known about TERT expression at the single-cell and single-molecule level. To address this, we analyzed TERT expression across 10 human cancer lines using single-molecule RNA fluorescent in situ hybridization (FISH) and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, spliced TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that spliced mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells.

Cytoplasmic SIRT1 inhibits cell migration and invasion by impeding epithelial-mesenchymal transition in ovarian carcinoma.

Sirtuin1 (SIRT1) is a mammalian NAD+-dependent type III deacetylase that plays paramount roles in diverse cellular processes. The nucleocytoplasmic shuttling of SIRT1 was discovered more than a decade ago, but the roles of subcellular SIRT1 localization in tumor progression remain unclear. Here, we report that cytoplasmic SIRT1 acts as a tumor suppressor in ovarian carcinoma. By creating ovarian carcinoma cell lines overexpressing wild-type SIRT1 and nuclear localization signals (NLSs) mutated SIRT1 together with both unbiased proteomic and acetylomic approaches and Transwell assays, we identified that mutations in the NLS sequences prevented SIRT1 from entering the nucleus, resulting in the predominant cytoplasmic localization of SIRT1; the cytoplasmic localization of SIRT1 suppressed the mesenchymal program, activated the epithelial program, and inhibited the migration and invasion of tumor cells, thus providing experimental evidence that SIRT1 functions as a tumor suppressor or oncogene may depend on its subcellular localization. Altogether, our findings may highlight a novel role of cytoplasmic SIRT1 in ovarian carcinoma, providing new possible insights for studies investigating the role of SIRT1 in tumor progression.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation.

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

Metformin promotes autophagy in ischemia/reperfusion myocardium via cytoplasmic AMPKα1 and nuclear AMPKα2 pathways.

AIMS: In myocardial ischemia-reperfusion (MI/R) injury, impaired autophagy function worsens cardiomyocyte death. AMP-activated protein kinase (AMPK) is a heterotrimeric protein that plays an important role in cardioprotection and myocardial autophagic function. AMPKα1 and α2 are localized primarily in the cytoplasm and nucleus, respectively, in cardiomyocytes, but the isoform-specific autophagy regulation of AMPK during MI/R remains unclear. MATERIALS AND METHODS: An MI/R model was built, and the protein expression of AMPKα1/α2, p-AMPK, mTOR, p-mTOR, TFEB, p-FoxO3a, SKP2, CARM1, TBP, Atg5, LAMP2, LC3B, and p62 during ischemia and reperfusion was determined by western blotting. Recombinant adeno-associated virus (serotype 9) vectors carrying tandem fluorescent-tagged LC3 or mRFP-GFP-LC3/GFP-LC3 were used to evaluate the autophagy status. AMPKα2 knockout mice were used for in vivo studies. KEY FINDINGS: Both cytoplasmic AMPKα1 and nuclear α2 subunit expression decreased during the reperfusion period, which led to AMPKα1-mTOR-TFEB and AMPKα2-Skp2-CARM1-TFEB signaling inhibition, respectively. The decreased TFEB level during reperfusion suppressed autophagy. Metformin could activate both the AMPKα1- and α2- mediated pathways, thus restoring autophagy flux during reperfusion. Nevertheless, in AMPKα2 knockout mice, nuclear α2-regulated Skp2-CARM1-TFEB signaling was inhibited, while α1-related signaling was comparatively unaffected, which partially impaired metformin-enhanced autophagy. SIGNIFICANCE: Our study suggests that metformin had the dual effects of promoting both cytoplasmic AMPKα1- and nuclear AMPKα2-related signaling to improve autophagic flux and restore cardiac function during MI/R.

Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1-mutant myelodysplastic syndromes as a therapeutic vulnerability.

Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.

Evolution and a revised nomenclature of P4 ATPases, a eukaryotic family of lipid flippases.

In all eukaryotic cells, P4 ATPases, also named phospholipid flippases, generate phospholipid asymmetry across biological membranes. This process is essential for cell survival, as it is required for vesicle budding and fusion in the secretory pathway. Several P4 ATPase isoforms can be identified in all sequenced eukaryotic genomes, but their evolution and interrelationships are poorly described. In this study, we conducted a thorough phylogenetic analysis of P4 ATPases in all major eukaryotic super-groups and found that they can be divided into three distinct families, P4A, P4B and P4C ATPases, all of which have an ancient origin. While P4B ATPases have been lost in plants, P4A ATPases are present in all eukaryotic super-groups. P4C ATPases form an intermediate group between the other two but appear to share a common origin with P4A ATPases. Sequence motifs unique to P4 ATPases are situated in the basal ATP hydrolyzing machinery. In addition, no clear signature motifs within P4 ATPase subgroups were found that could be related to lipid specificity, likely pointing to an elaborate transport mechanism in which different amino acid residue combinations in these pumps can result in recognition of the same substrate.