Putative Cofactor Research Articles

Crystallographic structure determination and analysis of a potential short-chain dehydrogenase/reductase (SDR) from multi-drug resistant Acinetobacter baumannii.

Bacterial antibiotic resistance remains an ever-increasing worldwide problem, requiring new approaches and enzyme targets. Acinetobacter baumannii is recognised as one of the most significant antibiotic-resistant bacteria, capable of carrying up to 45 different resistance genes, and new drug discovery targets for this organism is an urgent priority. Short-chain dehydrogenase/reductase enzymes are a large protein family with >60,000 members involved in numerous biosynthesis pathways. Here, we determined the structure of an SDR protein from A. baumannii and assessed the putative co-factor comparisons with previously co-crystalised enzymes and cofactors. This study provides a basis for future studies to examine these potential co-factors in vitro.

030 The Role of FasL-dependent Signaling in the Pathogenesis of Pemphigus Vulgaris

Pemphigus vulgaris is a chronic, life-altering bullous autoimmune disease with production of antibodies against the desmosomal proteins Desmogleins 3 and 1 (Dsg) causing acantholysis. Ubiquitous antibody deposition but discontinuous disease manifestation indicate the possible role of cofactors. In this study, we focus on FasL as a putative cofactor in PV-related acantholysis. To study cell-death dependent processes during PV, we used the spontaneously immortalized keratinocyte cell line HaCaT stimulated with AK23 (anti-Dsg3 ab) together with FasL as a cofactor.

035 Blocking IL-7, but not TLR7, signaling prevents the development of lupus-like autoimmunity in mice

Systemic lupus erythematosus (SLE) is a female-biased multisystem inflammatory disease with substantial morbidity and mortality and limited treatment options. Our lab previously found that VGLL3, a putative transcription co-factor with increased activity in female cells, can drive lupus-like systemic inflammation when overexpressed in mouse epidermis under the K5 promoter. IL-7 signaling was found to be elevated in the skin of lupus patients and K5-Vgll3+ mice alike. Our current results demonstrate that deleting peripheral Il7r in these mice prevents the development of disease phenotype, with amelioration of dermatitis, splenomegaly, lymphadenopathy, and kidney damage.

Abstract 2356: The epigenomic characterization of clear cell sarcoma

Abstract Clear cell sarcoma (CCS) is an aggressive malignancy that affects adolescents and young adults. CCS is a translocation-driven sarcoma subtype meaning a genetic fusion event defines and drives the malignancy. The chromosomal translocation occurring between EWSR1 on chromosome 22 and ATF1 on chromosome 12 results in the EWSR1-ATF1 fusion oncogene (hereafter abbreviated EA1) which drives more than 90% of CCS cases. The EA1 oncoprotein is a transcription factor that reprograms gene expression to drive CCS. However, the target genes of EA1 and how EA1 affects target gene expression levels are largely unknown. Elucidation of these effectors is critical in order to understand how the EA1 oncoprotein drives the epigenetic reprogramming in CCS. We utilized a robust mouse model that recapitulates CCS in which mice express TATCre-inducible EA1. Using CCS tumors that form in these mice, ChIP-sequencing identified where EA1 is binding along the genome. Analysis in combination with RNA-sequencing data revealed target genes that are bound by EA1 and either upregulated or downregulated compared to control tissue. Target gene expression levels were corroborated in a human context by RNA-sequencing of human tumors and a CCS human cell line. RNAi knockdown of EA1 in the human cell line confirmed functionality of the fusion protein at these target genes. We show that EA1 can bind at promoter regions to directly regulate transcription of target genes. These binding sites include the canonical ATF binding motif. The EA1 target genes overlap with known wildtype ATF1 target genes. Many upregulated genes are involved in cell cycle and proliferation as expected. Alternatively, EA1 can bind intergenic regions including H3K27ac-defined super enhancers to distally regulate target genes. These binding sites contain unique DNA motifs that are different from the canonical ATF binding motif by 1 base pair. One highly recurrent motif identified matches the motif of the AP1 complex, a putative co-factor that may be functioning with EA1. These so-called variant motifs appear to define enhancer regions where EA1 can bind along with AP1. In summary, EA1 binds the expected ATF motif at promoter regions to directly regulate target gene expression. Secondly, EA1 binds intergenic enhancer regions defined by variant motifs including the AP1 motif. Target genes can either be upregulated or downregulated by the fusion, suggesting that there are secondary epigenetic mechanisms. One such model involves differential co-factor composition, such as AP1 co-binding, that dictates where EA1 binds or subsequent transcriptional effects. This research sets up the foundation for how EA1 functions as a transcription factor including the distribution of EA1 across the genome, the motifs that EA1 binds, and the key genes that are regulated by EA1. These epigenetic findings are crucial to understanding how a powerful fusion oncoprotein drives this aggressive, under-studied malignancy. Citation Format: Benjamin B. Ozenberger, Li Li, Kevin B. Jones. The epigenomic characterization of clear cell sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2356.

Elucidation of an anaerobic pathway for metabolism of l-carnitine–derived γ-butyrobetaine to trimethylamine in human gut bacteria

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient l-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent l-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the l-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis We identify a set of genes up-regulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a putative flavin cofactor to catalyze a redox-neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from l-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of l-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

Architecture of the membrane-bound cytochrome c heme lyase CcmF.

The covalent attachment of one or multiple heme cofactors to their protein chain enables cytochromes c to be utilized in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH binding motifs. Here we report the three-dimensional structure of the heme lyase CcmF from Thermus thermophilus, a 643 aa integral membrane protein. CcmF contains a heme b cofactor at the bottom of a large cavity that opens towards the extracellular side to receive heme groups for cytochrome maturation from the heme chaperone CcmE. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.

Complicating Infections Associated with Common Endemic Human Respiratory Coronaviruses.

Coronaviruses OC43, 229E, NL63, and HKU1 are endemic human respiratory coronaviruses that typically cause mild to moderate upper respiratory infections, similar to the common cold. They also may cause simple and complicated lower respiratory infections, otitis media, asthma exacerbations, gastroenteritis, and a few systemic complications. These viruses are usually seasonal (with winter dominance) and affect nearly all age groups. The seasonal and annual variation in virus prevalence has implications for understanding the concept of acquired immunity and its persistence or diminution. Coronaviruses generally have outbreak potential in susceptible populations of any age, particularly in patients with comorbidities, who tend to have increased clinical disease. These 4 coronaviruses are often found in the context of what appears to be coinfection with other pathogens, but especially other viruses. If coronaviruses are not specifically tested for, the sole detection of a viral copathogen would suggest the pathogen is the causative agent, when a coronavirus may be culpable, or both. The detection of these viruses in circumstances where respiratory viruses are generally sought in clinical samples is, therefore, justified. These pathogens can be chronically shed from the respiratory tract, which is more likely to occur among immunocompromised and complicated patients. These viruses share the potential for genetic drift. The genome is among the largest of RNA viruses, and the capability of these viruses to further change is likely underestimated. Given the potential disease among humans, it is justified to search for effective antiviral chemotherapy for these viruses and to consider uses in niche situations should effective therapy be defined. Whereas SARS-CoV-2 may follow the epidemiological pattern of SARS-CoV and extinguish slowly over time, there is yet concern that SARS-CoV-2 may establish itself as an endemic human respiratory coronavirus similar to OC43, 2299E, NL63, and HKU1. Until sufficient data are acquired to better understand the potential of SARS-CoV-2, continued work on antiviral therapy and vaccination is imperative.

Sgd1 is an MIF4G domain-containing cofactor of the RNA helicase Fal1 and associates with the 5’ domain of the 18S rRNA sequence

ABSTRACTAssembly of eukaryotic ribosomal subunits is a complex and dynamic process involving the action of more than 200 trans-acting assembly factors. Although recent cryo-electron microscopy structures have provided information on architecture of several pre-ribosomal particles and the binding sites of many AFs, the RNA and protein interactions of many other AFs not captured in these snapshots still remain elusive. RNA helicases are key regulators of structural rearrangements within pre-ribosomal complexes and here we have analysed the eIF4A-like RNA helicase Fal1 and its putative cofactor Sgd1. Our data show that these proteins interact directly via the MIF4G domain of Sgd1 and that the MIF4G domain of Sgd1 stimulates the catalytic activity of Fal1 in vitro. The catalytic activity of Fal1, and the interaction between Fal1 and Sgd1, are required for efficient pre-rRNA processing at the A0, A1 and A2 sites. Furthermore, Sgd1 co-purifies the early small subunit biogenesis factors Lcp5 and Rok1, suggesting that the Fal1-Sgd1 complex likely functions within the SSU processome. In vivo crosslinking data reveal that Sgd1 binds to helix H12 of the 18S rRNA sequence and we further demonstrate that this interaction is formed by the C-terminal region of the protein, which is essential for its function in ribosome biogenesis.

Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis.

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R275 was present in a highly conserved PXRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity.IMPORTANCE Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness, and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmpX gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights into its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

The female-biased factor VGLL3 drives cutaneous and systemic autoimmunity.

Autoimmune disease is 4 times more common in women than men. This bias is largely unexplained. Female skin is "autoimmunity prone," showing upregulation of many proinflammatory genes, even in healthy women. We previously identified VGLL3 as a putative transcription cofactor enriched in female skin. Here, we demonstrate that skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash and systemic autoimmune disease that involves B cell expansion, autoantibody production, immune complex deposition, and end-organ damage. Excess epidermal VGLL3 drives a proinflammatory gene expression program that overlaps with both female skin and cutaneous lupus. This includes increased B cell-activating factor (BAFF), the only current biologic target in systemic lupus erythematosus (SLE); IFN-κ, a key inflammatory mediator in cutaneous lupus; and CXCL13, a biomarker of early-onset SLE and renal involvement. Our results demonstrate that skin-targeted overexpression of the female-biased factor VGLL3 is sufficient to drive cutaneous and systemic autoimmune disease that is strikingly similar to SLE. This work strongly implicates VGLL3 as a pivotal orchestrator of sex-biased autoimmunity.

Cul4 ubiquitin ligase cofactor DCAF12 promotes neurotransmitter release and homeostatic plasticity.

We genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. We suggest that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.

Microglial dynamics during brain development.

Microglial dynamics during brain development.

Prevention of Anaphylaxis Based on Risk Factors and Cofactors

Anaphylaxis is most commonly caused by drugs, food, and hymenoptera stings, or in certain instances may be idiopathic. A variety of risk factors, cofactors, and augmenting factors may act together to trigger anaphylaxis. Patients with certain underlying medical conditions may be at increased risk of anaphylaxis of certain etiologies, e.g., monoclonal mast cell activation syndromes and insect venom anaphylaxis, asthma, and fatal food-induced anaphylaxis especially in adolescents. Identification of risk factors, cofactors, and augmenting factors during clinical assessment is key to determining the type of in vitro and in vivo diagnostic tests that would be most appropriate in accurately identifying the putative allergen(s). Provocation tests, despite being the gold standard in allergy diagnosis, should be avoided in anaphylaxis as the risks outweigh the benefits. Accurate diagnosis of the putative allergen and cofactor is key to both prevention of recurrence of anaphylaxis, avoidance measures, and interventions like allergen-specific immunotherapy, which may impact the quality of life of allergy sufferers and their family members.

Detection of Torque Teno Sus Virus in Pork Bile and Liver Sausages.

Torque teno viruses (TTV) are small DNA viruses widespread among humans and pigs. The clinical significance of TTV infections in either humans or pigs is uncertain. In fact, TTV viremia is highly prevalent in patients with different pathologies, but it can also be frequently observed in healthy subjects. Virus infection in pigs is considered a putative cofactor in several diseases; despite being detected frequently in healthy animals, its role still remains unknown. The present study aimed to investigate the presence of Torque teno sus virus (TTSuV) in 62 bile samples collected from pigs at slaughterhouse and in 36 fresh pork liver sausages bought at point of sale. Quantitative Real-Time PCR, confirmed that 19.4 and 58.3% of bile and sausage samples tested positive for TTSuV, respectively. The mean viral load was established as 5.6×104GE/µl for bile and 7.16×103GE/g for sausages. TTSuV nucleotide sequence analysis confirmed a wide heterogeneity among the circulating TTSuV strains, which included both TTSuV1 and TTSuV2.

Identification of biotransformation enzymes in the antennae of codling moth Cydia pomonella

Biotransformation enzymes are found in insect antennae and play a critical role in degrading xenobiotics and odorants. In Cydia pomonella, we identified 26 biotransformation enzymes. Among these enzymes, twelve carboxylesterases (CXEs), two aldehyde oxidases (AOXs) and six alcohol dehydrogenases (ADs) were predominantly expressed in antennae. Each of the CpomCXEs presents a conserved catalytic triad “Ser-His-Glu”, which is the structural characteristic of known insect CXEs. CpomAOXs present two redox centers, a FAD-binding domain and a molybdenum cofactor/substrate-binding domain. The antennal CpomADs are from two protein families, short-chain dehydrogenases/reducetases (SDRs) and medium-chain dehydrogenases/reducetases (MDRs). Putative catalytic active domain and cofactor binding domain were found in these CpomADs. Potential functions of these enzymes were determined by phylogenetic analysis. The results showed that these enzymes share close relationship with odorant degrading enzymes (ODEs) and resistance-associated enzymes of other insect species. Because of commonly observed roles of insect antennal biotransformation enzymes, we suggest antennal biotransformation enzymes presented here are candidate that involved in degradation of odorants and xenobiotics within antennae of C. pomonella.

Host determinants of HTLV-1 integration site preference

Integration of the provirus into the host genome is an essential step in the establishment of a chronic retroviral infection. Retroviruses do not integrate at random. Each genus of retrovirus preferentially integrates in genomic sites associated with certain genomic features. This preference is thought to result from an interaction of the pre-integration complex with a specific host co-factor. Such an interaction has been well documented between HIV-1 integrase and lens epithelium-derived growth factor (LEDGF) and between MLV and members of the Bromoextraterminal Domain Protein (BET) family. HTLV-1 also has a reproducible preference to integrate near various genomic features; however, the putative host co-factor of HTLV-1 integrase remains unknown. We present evidence that demonstrates that the HTLV-1 integrase co-factor may be the protein phosphatase 2A (PP2A) complex. Two independent protein interaction screens (yeast-2-hybrid and co-immunoprecipitation) both revealed an interaction between HTLV-1 integrase and the PP2A holoenzyme. We used co-culture with HTLV-1 producer cell line MT2 to infect rat cell lines containing an inducible knockdown of PP2A. We found that PP2A-knockdown altered the integration profile of these de novo integration sites. In contrast to infection in PP2A-competent cells, integration sites in PP2A-knockdown cells lacked the characteristic proximity to genomic features associated with transcriptional activity (genes, transcription start sites, CpG islands) and to epigenetic marks associated with active chromatin. We are in the process of investigating how PP2A inhibitors and shRNAs targeting specific regulatory subunits of the PP2A holoenzyme influence integration site selection.

Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

Abstract LB-87: Analysis of JAZF1 loss-of-function reveals its role in regulation of genes relevant for prostate cancer

Abstract Genome-wide association studies (GWAS) have mapped one of the prostate cancer susceptibility regions to a single nucleotide polymorphism (SNP) rs10486567 within intron 2 of the gene that encodes JAZF1, a putative transcriptional co-factor. In order to translate GWAS findings into clinically relevant information that will ultimately enable us to improve the management of prostate cancer it is essential to identify the biological role of the susceptibility variant in cells and tissues. To date, only a few studies address the biological function of JAZF1 and none in prostate cancer. To investigate the function of JAZF1 we generated expression profiles of several prostate cancer cell lines, in which JAZF1 was silenced. We conducted RNA-Seq of two metastatic prostate cancer cell lines, LNCaP and DU145, in which siRNA-mediated RNAi had been employed to induce JAZF1 loss-of-function (LOF). For this study, three different JAZF1 siRNAs were used, along with a siRNA (siNeg) serving as control for transfection-related changes in gene expression. Approximately 1,500 genes were identified as deregulated in both cell lines. Computational analysis of these genes showed enrichment for genes associated with prostate cancer (p=2.4x10-6, Ingenuity Pathway Analysis), including MAP2K4 and MET. These two genes were up-regulated following JAZF1 LOF in both cell lines, consistent with JAZF1's putative role as a transcriptional repressor. Among the genes down-regulated following siRNA silencing of JAZF1was ID1. ID1 is a negative transcriptional regulator that functions as part of the mitotic checkpoint machinery. One of ID1's target genes is the cell-cycle inhibitor p21. In JAZF1-silenced DU145 cells we observed a decrease in ID1 mRNA expression that was correlated with an increase in CDKN1A expression, the gene encoding p21; an observation confirmed at a protein level. Examination of publically available prostate cancer gene expression data indicated a correlation between JAZF1 and ID1 expression (p<0.001). To further analyze the putative regulation of ID1 by JAZF1, and thus p21, we used lentivirus-mediated delivery of JAZF1-shRNAs to stably transduce DU145 cells,, OPCN-1 non-tumorigenic cells, and a non-metastatic prostate tumor epithelium cell line, OPCT-1. Under conditions of long-term JAZF1 LOF we observed increased ID1 expression as measured by Western Blot analysis and concurrent decreases in, p21 protein levels in all three-cell lines. Although this result appears contradictory to our initial observation of siRNA-mediated JAZF1 silencing, this difference may reflect cell line and temporal specific activity of JAZF1 as a transcriptional activator or repressor of ID1 expression. These findings are consistent though with JAZF1 functioning as a regulator of ID1, a protein reported to be over-expressed in prostate cancer that is associated with cancer cell proliferation and metastasis formation through activation of various oncogenic pathways. Citation Format: Helen Gharwan, Tamara Jones, Wei Tang, Stefan Ambs, Ludmilla Prokunina, Natasha Caplen. Analysis of JAZF1 loss-of-function reveals its role in regulation of genes relevant for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2014-LB-87

The MoxR ATPase RavA and Its Cofactor ViaA Interact with the NADH:Ubiquinone Oxidoreductase I in Escherichia coli

MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37°C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I). Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.