Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.     

Iron Regulatory Protein-1 Protects against Mitoferrin-1-deficient Porphyria

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1^+/gt;Irp1^−/− erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5′-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1^gt/gt cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1^gt/gt cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.     

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.Members of the Archaea produce many proteins that require iron-sulfur cluster cofactors, including redox proteins, aconitase-like dehydratases, radical S-adenosylmethionine enzymes, and RNA polymerase (9, 13, 18, 32). Methanogenic archaea are obligate anaerobes, and many heterotrophic archaea grow anaerobically, indicating that oxidative stress has not limited the proliferation of iron-sulfur proteins in these lineages. Archaea must have a mechanism to assemble Fe-S clusters, but many members lack homologs of the known bacterial and eukaryotic Nif or Isc systems, suggesting that an alternative system is present (see Table S1 in the supplemental material) (14, 15).Some euryarchaea have homologs of the bacterial genes iscS (encoding cysteine desulfurase) and iscU (encoding a scaffold protein). Yet many other archaea, including the euryarchaea Pyrococcus furiosus, Methanocaldococcus jannaschii, and Methanococcus maripaludis, plus most crenarchaea, either lack a homologous cysteine desulfurase gene or have no homologs of A-type or U-type scaffold genes. Due to their sulfide-rich environments, it is reasonable that the anaerobic archaea may use an inorganic sulfur source to assemble Fe-S clusters. Most archaeal genome sequences do carry homologs of the sufBC genes that are part of the alternative Suf system for Fe-S cluster biosynthesis (36). Biochemical studies have shown that SufC is an ABC-type ATPase and that SufB is a persulfide acceptor that may act as a site for Fe-S cluster assembly (20). The SufB and SufC proteins interact, and SufB stimulates the ATPase activity of SufC. We hypothesize that the Archaea share a common mechanism for Fe-S cluster biosynthesis, supplemented with genes acquired by horizontal gene transfer in some lineages.A screen for Salmonella enterica bacteria defective in thiamine biosynthesis identified lesions in the apbC locus (28) that compromised Fe-S metabolism (33). An abpC mutant cannot grow with tricarballylate as a carbon and energy source, which may be due to a defect in assembling or repairing [4Fe-4S] clusters in the membrane-bound TcuB protein (21, 22). ApbC is a 40-kDa cytoplasmic protein with Walker A and B nucleotide-binding domains and two conserved carboxy-terminal cysteine residues separated by two amino acids (Cys-X-X-Cys). Mutational analyses have shown that ApbC proteins with directed changes in the Cys-X-X-Cys or Walker A motifs are not active in vivo (6). Suppressor analysis allowed the conclusion that a degree of functional redundancy between ApbC and the Fe-S scaffold protein IscU exists (4, 38). Although purified ApbC does not contain iron or sulfur, biochemical studies showed that ApbC can bind an Fe-S cluster and rapidly transfer it to an apoprotein (5).It is thought that in eukaryotes, Fe-S clusters are assembled by the mitochondrial iron-sulfur cluster (ISC) system (23). The clusters are transported into the cytosol and delivered by the cytosolic iron-sulfur protein assembly system. Two components of this system, Nbp35 and Cfd1, are homologs of bacterial ApbC (Fig. ​(Fig.1)1) and act as intermediate Fe-S cluster-trafficking proteins in the cytosol (16, 27, 30). Electron paramagnetic resonance, Mössbauer, and absorbance spectra of the Saccharomyces cerevisiae, human, and Arabidopsis Nbp35 holoproteins suggest that these holoproteins form dimers with stable amino-terminal [4Fe-4S] clusters and a shared carboxy-terminal [4Fe-4S] cluster (10, 34).Open in a separate windowFIG. 1.A protein sequence alignment of bacterial, archaeal, and eukaryotic ApbC/Nbp35 homologs was constructed using the ClustalW program (version 1.83) (37). The sequence of the S. enterica serovar Typhimurium protein (ApbC; RefSeq accession no. NP_461098.1) is shown without the amino-terminal domain that is not homologous to the amino-terminal domains of the archaeal and eukaryotic proteins. The archaeal homologs are from S. solfataricus (SSO0460; accession no. NP_341994.1), P. furiosus (PF1145; accession no. NP_578874.1), Methanosarcina acetivorans (MA4246; accession no. NP_619111.1), M. jannaschii (MJ0283; accession no. NP_247256.1), and M. maripaludis (MMP0704; accession no. NP_987824.1). The two paralogs from S. cerevisiae are Nbp35 (accession no. NP_011424.1) and Cfd1 (accession no. NP_012263.1). Conserved amino acid residues are shown in white on a black background. Similar residues are shown in black on a gray background. The four conserved amino-terminal cysteine residues shared by the MMP0704 and Nbp35p proteins are boxed. Asterisks above the sequences indicate MMP0704 residues replaced by mutagenesis in this study. A vertical bar indicates the N termini of the truncated proteins MJ0283(19-290) and MMP0704(20-289).Archaeal homologs of bacterial ApbC and eukaryotic Nbp35 are underannotated as nucleotide-binding proteins or misannotated as cobyrinic acid a,c-diamide synthases in sequence databases. The hallmarks of the Nbp35 sequences are an amino-terminal ferredoxin-like domain, an ATP-binding motif, and two conserved carboxy-terminal cysteine residues that are believed to bind an Fe-S cluster. The amino-terminal ferredoxin-like domain is absent in the ApbC family of proteins. The ApbC and Nbp35 proteins belong to a large superfamily of P-loop-containing nucleoside triphosphate hydrolases that also includes the bacterial MinD and CooC proteins. The M. maripaludis MMP0704 protein shows approximately 40% amino acid identity to both the S. enterica ApbC and S. cerevisiae Nbp35 proteins (Fig. ​(Fig.1).1). However, the MMP0704 protein also shows 30% sequence identity to two paralogous proteins from M. maripaludis. The genome sequence of M. maripaludis encodes at least nine paralogs, although only the MMP0704 protein contains the conserved cysteine residues found in most ApbC/Nbp35 proteins.The experiments described herein identified the first archaeal proteins that form functional Fe-S carrier proteins. The apbC/NBP35 homologs from M. maripaludis (MMP0704), M. jannaschii (MJ0283), and Sulfolobus solfataricus (SSO0460) allowed an S. enterica strain with an apbC null mutation to grow on tricarballylate. Genetic studies showed that the Walker A box and at least one cysteine residue from the Cys-X-X-Cys motif were required for in vivo functionality. The unique amino-terminal ferredoxin-like domains of the MMP0704 and MJ0283 proteins were not required. Purified MMP0704 proteins bound Fe-S clusters. Orthologs of ApbC/Npb35 proteins were found in all of the available genomes of free-living archaea, identifying this protein family as an ancient part of the Fe-S assembly system that evolved before the divergence of Archaea and Eukarya.     

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from l-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2′-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5′-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess l-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.     

Role for Heat Shock Protein 90α in the Proliferation and Migration of HaCaT Cells and in the Deep Second-Degree Burn Wound Healing in Mice

Inflammation, proliferation, and tissue remodeling are essential steps for wound healing. The hypoxic wound microenvironment promotes cell migration through a hypoxia—heat shock protein 90 alpha (Hsp90α)—low density lipoprotein receptor-related protein-1 (LRP-1) autocrine loop. To elucidate the role of this autocrine loop on burn wound healing, we investigated the expression profile of Hsp90α at the edge of burn wounds and found a transient increase in both mRNA and protein levels. Experiments performed with a human keratinocyte cell line—HaCaT also confirmed above results. 17-dimethylaminoethylamino-17demethoxygeldanamycin hydrochloride (17-DMAG), an Hsp90α inhibitor, was used to further evaluate the function of Hsp90α in wound healing. Consistently, topical application of Hsp90α in the early stage of deep second-degree burn wounds led to reduced inflammation and increased tissue granulation, with a concomitant reduction in the size of the wound at each time point tested (p<0.05). Consequently, epidermal cells at the wound margin progressed more rapidly causing an expedited healing process. In conclusion, these results provided a rationale for the therapeutic effect of Hsp90α on the burn wound management.     

Novel DNA Variants and Mutation Frequencies of hMLH1 and hMSH2 Genes in Colorectal Cancer in the Northeast China Population

Research on hMLH1 and hMSH2 mutations tend to focus on Lynch syndrome (LS) and LS-like colorectal cancer (CRC). No studies to date have assessed the role of hMLH1 and hMSH2 genes in mass sporadic CRC (without preselection by MSI or early age of onset). We aimed to identify novel hMLH1 and hMSH2 DNA variants, to determine the mutation frequencies and sites in both sporadic and LS CRC and their relationships with clinicopathological characteristics of CRC in Northeast of China. 452 sporadic and 21 LS CRC patients were screened for germline and somatic mutations in hMLH1 and hMSH2 genes with PCR–SSCP sequencing. We identified 11 hMLH1 and seven hMSH2 DNA variants in our study cohort. Six of them were novel: four in hMLH1 gene (IVS8-16 A>T, c.644 GAT>GTT, c.1529 CAG>CGG and c.1831 ATT>TTT) and two in hMSH2 gene (−39 C>T, insertion AACAACA at c.1127 and deletion AAG at c.1129). In sporadic CRC, germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 15.59% and 17.54%, respectively (p = 0.52). Germline mutations present in hMLH1 and hMSH2 genes were 5.28% and 10.78%, respectively (p<0.01). Somatic mutations in hMLH1 and hMSH2 genes were 6.73% and 11.70%, respectively (p = 0.02). In LS CRC, both germline and somatic mutation frequencies of hMLH1/hMSH2 gene were 28.57%. The most prevalent germline mutation site in hMSH2 gene was c.1168 CTT>TTT (3.90%), a polymorphism. Somatic mutation frequency of hMLH1/hMSH2 gene was significantly different in proximal, distal colon and rectal cancer (p = 0.03). Our findings elucidate the mutation spectrum and frequency of hMLH1 and hMSH2 genes in sporadic and LS CRC, and their relationships with clinicopathological characteristics of CRC.     

Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB)2

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL₂ bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)₂ complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)₂ complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)₂ crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)₂(ChlL₂)₂ complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.     

Escherichia coli RIC Is Able to Donate Iron to Iron-Sulfur Clusters

Escherichia coli RIC (Repair of Iron Centers) is a diiron protein previously reported to be involved in the repair of iron-sulfur proteins damaged by oxidative or nitrosative stresses, and proposed to act as an iron donor. This possible role of RIC was now examined specifically by evaluating its ability to donate iron ions to apo-iron-sulfur proteins, determining the iron binding constants and assessing the lability of its iron ions. We show, by UV-visible, EPR and resonance Raman spectroscopies that RIC may participate in the synthesis of an iron-sulfur cluster in the apo-forms of the spinach ferredoxin and IscU when in the presence of the sulfide donating system IscS and L-cysteine. Iron binding assays allowed determining the as-isolated and fully reduced RIC dissociation constants for the ferric and ferrous iron of 10⁻²⁷ M and 10⁻¹³ M, respectively. Mössbauer studies revealed that the RIC iron ions are labile, namely when the center is in the mixed-valence redox form as compared with the (μ-oxo) diferric one. Altogether, these results suggest that RIC is capable of delivering iron for the formation of iron-sulfur clusters.     

Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L^-1 to 5.5 mg L^-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.     

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative λ_max at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S₈ configuration. No labeled elemental sulfur was detected when the cells were grown on [³⁵S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.     

Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.     

The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.     

Therapeutic Benefits of Induced Pluripotent Stem Cells in Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive increases in vascular resistance and the remodeling of pulmonary arteries. The accumulation of inflammatory cells in the lung and elevated levels of inflammatory cytokines in the bloodstream suggest that inflammation may play a role in PAH. In this study, the benefits of induced pluripotent stem cells (iPSCs) and iPSC-conditioned medium (iPSC CM) were explored in monocrotaline (MCT)-induced PAH rats. We demonstrated that both iPSCs and iPSC CM significantly reduced the right ventricular systolic pressure and ameliorated the hypertrophy of the right ventricle in MCT-induced PAH rats in models of both disease prevention and disease reversal. In the prevention of MCT-induced PAH, iPSC-based therapy led to the decreased accumulation of inflammatory cells and down-regulated the expression of the IL-1β, IL-6, IL-12α, IL-12β, IL-23 and IFNγ genes in lung specimens, which implied that iPSC-based therapy may be involved in the regulation of inflammation. NF-κB signaling is essential to the inflammatory cascade, which is activated via the phosphorylation of the NF-κB molecule. Using the chemical inhibitor specifically blocked the phosphorylation of NF-κB, and in vitro assays of cultured human M1 macrophages implied that the anti-inflammation effect of iPSC-based therapy may contribute to the disturbance of NF-κB activation. Here, we showed that iPSC-based therapy could restore the hemodynamic function of right ventricle with benefits for preventing the ongoing inflammation in the lungs of MCT-induced PAH rats by regulating NF-κB phosphorylation.     

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn''s activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn''s dynamics at the PM and its relation to its inhibition of exocytosis.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.