CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes.

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Spatial organization of bacterial sphingolipid synthesis enzymes.

Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death, angiogenesis, and inflammation. While it was previously believed that sphingolipids were quite rare among bacteria, bioinformatic analysis of the recently identified bacterial sphingolipid synthesis genes suggests that these lipids are likely to be produced by a wide range of microbial species. The sphingolipid synthesis pathway consists of three critical enzymes. Serine palmitoyltransferase catalyzes the condensation of serine with palmitoyl-CoA (or palmitoyl-acyl carrier protein), ceramide synthase adds the second acyl chain, and a reductase reduces the ketone present on the long-chain base. While there is general agreement regarding the identity of these bacterial enzymes, the precise mechanism and order of chemical reactions for microbial sphingolipid synthesis is more ambiguous. Two mechanisms have been proposed. First, the synthesis pathway may follow the well characterized eukaryotic pathway in which the long-chain base is reduced prior to the addition of the second acyl chain. Alternatively, our previous work suggests that addition of the second acyl chain precedes the reduction of the long-chain base. To distinguish between these two models, we investigated the subcellular localization of these three key enzymes. We found that serine palmitoyltransferase and ceramide synthase are localized to the cytoplasm, whereas the ceramide reductase is in the periplasmic space. This is consistent with our previously proposed model wherein the second acyl chain is added in the cytoplasm prior to export to the periplasm where the lipid molecule is reduced.

Sequence-specific targeting of Caenorhabditis elegans C-Ala to the D-loop of tRNAAla.

Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.

Type 2 transglutaminase in the nucleus: the new epigenetic face of a cytoplasmic enzyme.

One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.

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.

Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP).

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.

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.

Genome-wide survey of the phosphofructokinase family in cassava and functional characterization in response to oxygen-deficient stress.

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.

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.

Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases.

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.

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.

A conserved sequence motif bridges two protein kinases for enhanced phosphorylation and nuclear function of a splicing factor.

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans.

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion.

In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 µM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.

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.