Direct Protein-protein Interaction Research Articles

Reversible SAHH inhibitor protects against glomerulonephritis in lupus-prone mice by downregulating renal \u03b1-actinin-4 expression and stabilizing integrin-cytoskeleton linkage

BackgroundGlomerulonephritis is one of the major complications and causes of death in systemic lupus erythematosus (SLE) and is characterized by glomerulosclerosis, interstitial fibrosis, and tubular atrophy, along with severe persistent proteinuria. DZ2002 is a reversible S-adenosyl-l-homocysteine hydrolase (SAHH) inhibitor with potent therapeutic activity against lupus nephritis in mice. However, the molecular events underlying the renal protective effects of DZ2002 remained unclear. This study is designed to uncover the molecular mechanisms of DZ2002 on glomerulonephritis of lupus-prone mice.MethodsWe conducted a twice-daily treatment of DZ2002 on the lupus-prone NZB/WF1 mice, and the progression of lupus nephritis and alteration of renal function were monitored. The LC-MS-based label-free quantitative (LFQ) proteomic approach was applied to analyze the kidney tissue samples from the normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. KEGG pathway enrichment and direct protein-protein interaction (PPI) network analyses were used to map the pathways in which the significantly changed proteins (SCPs) are involved. The selected proteins from proteomic analysis were validated by Western blot analysis and immunohistochemistry in the kidney tissues.ResultsThe twice-daily regimen of DZ2002 administration significantly ameliorated the lupus nephritis and improved the renal function in NZB/WF1 mice. A total of 3275 proteins were quantified, of which 253 proteins were significantly changed across normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. Pathway analysis revealed that 13 SCPs were involved in tight junction and focal adhesion process. Further protein expression validation demonstrated that DZ2002-treated NZB/WF1 mice exhibited downregulation of α-actinin-4 and integrin-linked kinase (ILK), as well as the restoration of β1-integrin activation in the kidney tissues compared with the vehicle-treated ones.ConclusionsOur study demonstrated the first evidence for the molecular mechanism of SAHH inhibitor on glomerulonephritis in SLE via the modulation of α-actinin-4 expression and focal adhesion-associated signaling proteins in the kidney.

Identification of Heteroreceptors Complexes and Signal Transduction Events Using Bioluminescence Resonance Energy Transfer (BRET).

Detecting protein-protein interactions by co-immunoprecipitation provided a major advancement in the immunology research field. In the G-protein-coupled receptors (GPCRs) research field, colocalization and co-immunoprecipitation were used to detect interactions, but doubts arose due to specificity of the antibodies (monoclonal in the case of receptors related to immunology and polyclonal in the case of GPCRs) and due to the possibility of false positive due to the potential occurrence of bridging proteins. Accordingly, new methodological approaches were needed, and energy transfer techniques have been instrumental to detect direct protein-protein, protein-receptor or receptor-receptor interactions. Of the two most relevant methods (Förster, or fluorescence resonance energy transfer: FRET and Bioluminescence energy transfer: BRET), the protocol for BRET is here presented. BRET has been instrumental to detect direct interactions between GPCRs and has contributed to demonstrate that GPCR dimers/oligomer functionality is different from that exerted by individual receptors. Advantages outweigh those of FRET as no fluorescence source is needed. Interestingly, BRET is not only useful to validate interactions detected by other means or hypothesized in the basis of indirect evidence, but to measure signal transduction events. In fact, BRET may, for instance, be used to assess β-arrestin recruitment to activated GPCRs.

Emerging Mechanisms of Cellular Iron Transport and Trafficking

Iron is an essential nutrient required by every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mono- and dinuclear iron centers) are involved in virtually every major metabolic process in the cell. Iron deficiency continues to be the most common nutritional deficiency in the world, while iron overload is a feature of an increasing number of human diseases, including genetic disorders such as hereditary hemochromatosis, thalassemias, and Friedreich ataxia, as well as chronic inflammatory diseases of the liver, such as hepatitis C and steatohepatitis. The biochemical mechanisms by which iron causes toxicity are not completely known. Previously, we identified Poly rC-binding protein 1 (Pcbp1) as a protein that directly binds and delivers iron to ferritin, the major iron storage protein in mammalian cells. Pcbp1 and its paralog Pcbp2 are multifunctional adaptors that bind cellular RNA, DNA, proteins, and iron, altering the fate of their binding partners. Ferritin is a large polypeptide, containing 24 subunits of H- and L-chains assembled into a hollow sphere. Reduced (ferrous) iron enters the sphere through pores formed between the subunits and is oxidized on the interior surface to form nanocrystals of ferric oxyhydroxides. Pcbp1 is the first example of an iron chaperone- a protein that specifically binds iron ions and delivers them to target proteins, such as ferritin, through direct protein-protein interactions. In cultured cells, Pcbp1 is also required for efficient iron delivery to non-heme enzymes in addition to ferritin. These enzymes include the mononuclear iron-containing prolyl hydroxylases that regulate HIF and the dinuclear iron-containing deoxyhypusine hydroxylase, which is required for the modification of lysine to hypusine. In the adult human, 70% of total body iron is present in circulating erythrocytes, which are produced by the bone marrow at a rate of 2 million reticulocytes per second. We examined the roles of Pcbp1 and Ncoa4 in this extraordinary flux of iron through the erythron. Ncoa4 is the cargo receptor that recruits ferritin into the autophagosome for degradation in the lysosome. Using an in vitro model of erythroid differentiation, we showed that depletion of Pcbp1 or Ncoa4 impeded trafficking of iron through ferritin, which impaired the synthesis of heme and hemoglobin. Mice with tamoxifen-induced Pcbp1 deficiency exhibited a microcytic anemia typical of iron deficiency, with activation of compensatory erythropoiesis. The role of ferritin in erythropoiesis has been controversial, but our studies indicate that iron flux through ferritin is an obligatory process in the early stages of erythroid terminal differentiation. Our in vitro studies of erythrocytes revealed that the interactions of Pcbp1 and Ncoa4 with ferritin changed during differentiation, with maximal binding of Pcbp1 at early stages and Ncoa4 at late stages. These binding activities are regulated by cellular iron in mechanistically distinct ways. Ongoing studies of Pcbp1 deficiency in other murine tissues suggest the importance of iron chaperones in maintaining the bioavailable pool of iron in mammalian cells. DisclosuresNo relevant conflicts of interest to declare.

Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein.

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

A novel fatty acid-binding protein 5-estrogen-related receptor α signaling pathway promotes cell growth and energy metabolism in prostate cancer cells.

Epidermal or cutaneous fatty acid-binding protein is an intracellular lipid-binding protein, also known as FABP5, and its expression level is closely related to cancer cell proliferation and metastatic activities in various types of carcinoma. However, the molecular mechanisms of FABP5 in cancer cell proliferation and its other functions have remained unclear. In the present study, we have clearly revealed that FABP5 activated expression of metabolic genes (ATP5B, LCHAD, ACO2, FH and MFN2) via a novel signaling pathway in an ERRα (estrogen-related receptor α)-dependent manner in prostate cancer cell lines. To clarify the novel function of FABP5, we examined the activation mechanisms of the ERRα target genes via FABP5. A direct protein-protein interaction between FABP5 and ERRα was demonstrated by immunoprecipitation and GST pull-down assays. We have clearly revealed that FABP5 interacted directly with transcriptional complex containing ERRα and its co-activator PGC-1β to increase expression of the ERRα target genes. In addition, we have shown that FABP5 knockdown induced high energy stress leading to induction of apoptosis and cell cycle arrest via AMPK-FOXO3A signaling pathway in prostate cancer cells, suggesting that FABP5 plays an important role in cellular energy status directing metabolic adaptation to support cellular proliferation and survival.

Abstract A48: MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC

Abstract Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and the molecular heterogeneity within TNBC heightens the challenge of developing effective targeted therapies. Receptor tyrosine kinases (RTKs), in particular MET and EGFR, are promising therapeutic targets for TNBC due to their high expression in multiple molecular TNBC subtypes and the tendency for cancers to become “kinase addicted.” We and others have demonstrated that MET is highly expressed in TNBC and its expression correlates with poor prognosis. EGFR expression is also elevated in up to 72% of TNBCs and correlates with poor prognosis in TNBC patients. Recently, we demonstrated that MET and EGFR are coordinately and highly expressed across all TNBC subtypes and the efficacy of combined MET and EGFR inhibition in TNBC patient-derived xenograft (PDX) models. Even though MET and EGFR receptors are actionable targets due to their high activity in TNBC, crosstalk between MET and EGFR has been implicated in therapeutic resistance to kinase inhibitors in several cancer types and needs to be evaluated in TNBC. There is strong evidence demonstrating the critical role of redundant RTK signaling networks in resistance to tyrosine kinase inhibitors (TKIs). Specifically, crosstalk between MET and EGFR has been implicated in therapeutic resistance to EGFR or MET inhibitors in colon, gastric, and lung cancers. Since MET and EGFR are highly expressed in a substantial proportion of TNBCs, we used TNBC PDX models and TNBC cell lines to interrogate mechanisms of resistance to MET and EGFR kinase inhibition. We hypothesized that coexpression, interaction, and activation of MET and EGFR promote acquired TKI resistance and an adaptive kinome response in TNBC. Using TNBC PDX models that highly express both MET and EGFR, we evaluated MET and EGFR activity, localization, and interaction after 3 days or 3 weeks of treatment with MET (glesatinib, crizotinib) and/or EGFR inhibitors (erlotinib). After 3 weeks of dual MET and EGFR inhibition in TNBC PDXs, only a small population of “resistant” cells remained. In these resistant populations, we observed increased MET and EGFR colocalization and increased MET and EGFR activation. To understand how colocalization of MET and EGFR promotes signaling redundancy and crosstalk, we asked whether MET and EGFR directly interact in TNBC PDX using a proximity ligation assay (PLA). PLA uses antibody specificity to detect direct protein-protein interactions at physiologic levels in vitro and in vivo. We observed a significant increase in MET EGFR interactions only in resistant cell populations that had been treated with both MET and EGFR inhibitors. AKT and ERK signaling were also increased in TKI-resistant cells, indicating that these are key survival pathways in resistance. Interestingly, we did not observe an increase in MET-EGFR activity or interaction in PDX tumors treated for only 3 days. Even though TNBC cells (HCC70) showed increased colocalization of MET and EGFR with combined glesatinib and erlotinib treatment, there was not a significant increase in MET-EGFR interactions. Evaluation of MET-EGFR interaction in primary TNBC patient samples revealed significant MET-EGFR interactions in a MET-amplified tumor and lymph node metastases. To our knowledge, this is the first time MET-EGFR interactions have been observed in TNBC in vivo and in human patients. Moreover, MET-EGFR interactions are able to maintain MET, EGFR, ERK, and AKT activity through a yet-unknown mechanism. These results imply that MET EGFR interaction may be a unique mechanism of resistance to kinase inhibition that may be inherent in human TNBCs with genomic amplification of MET or EGFR. Citation Format: Elizabeth Tovar, Curt Essenburg, Anderson Peck, Lisa Turner, Zachary Madaj, Matthew Smith, James Christensen, Marianne Melnik, Eric Haura, Matthew Steensma, Carrie Graveel. MET and EGFR interaction promotes acquired resistance to kinase inhibition in TNBC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A48.

Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato.

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome-wide association study (GWAS) on type-I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map-based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc-finger protein. Transgenic experiments showed that hair-absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type-I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein-protein interaction between them was further detected through pull-down and yeast two-hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.

The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201.

Two almost identical gene clusters (phz1 and phz2) are responsible for phenazine-1-carboxylic acid (PCA) production in Pseudomonas aeruginosa (P. aeruginosa) strain MSH (derived from strain PA1201). Here, we showed that the anti-activator QslA negatively regulated PCA biosynthesis and phz1 expression in strain PA1201 but had little effect on phz2 expression. This downregulation was mediated by a 56-bp region within the 5′-untranslated region (5′-UTR) of the phz1 promoter and was independent of LasR and RsaL signaling. QslA also negatively regulated Pseudomonas quinolone signal (PQS) production. Indeed, QslA controlled the PQS threshold concentration needed for PQS-dependent PCA biosynthesis. The quorum sensing regulator MvfR was required for the QslA-dependent inhibition of PCA production. We identified a direct protein–protein interaction between QslA and MvfR. The ligand-binding domain of MvfR (residues 123–306) was involved in this interaction. Our results suggested that MvfR bound directly to the promoter of the phz1 cluster. QslA interaction with MvfR prevented the binding of MvfR to the phz1 promoter regions. Thus, this study depicted a new mechanism by which QslA controls PCA and PQS biosynthesis in P. aeruginosa.

An ETHYLENE RESPONSE FACTOR-MYB Transcription Complex Regulates Furaneol Biosynthesis by Activating QUINONE OXIDOREDUCTASE Expression in Strawberry.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone is a major contributor to the aroma of strawberry (Fragaria × ananassa) fruit, and the last step in its biosynthesis is catalyzed by strawberry quinone oxidoreductase (FaQR). Here, an ethylene response factor (FaERF#9) was characterized as a positive regulator of the FaQR promoter. Linear regression analysis indicated that FaERF#9 transcript levels were correlated significantly with both FaQR transcripts and furanone content in different strawberry cultivars. Transient overexpression of FaERF#9 in strawberry fruit significantly increased FaQR expression and furaneol production. Yeast one-hybrid assays, however, indicated that FaERF#9 by itself did not bind to the FaQR promoter. An MYB transcription factor (FaMYB98) identified in yeast one-hybrid screening of the strawberry cDNA library was capable of both binding to the promoter and activating the transcription of FaQR by ∼5.6-fold. Yeast two-hybrid assay and bimolecular fluorescence complementation confirmed a direct protein-protein interaction between FaERF#9 and FaMYB98, and in combination, they activated the FaQR promoter 14-fold in transactivation assays. These results indicate that an ERF-MYB complex containing FaERF#9 and FaMYB98 activates the FaQR promoter and up-regulates 4-hydroxy-2,5-dimethyl-3(2H)-furanone biosynthesis in strawberry.

Loss of PDK4 switches the hepatic NF-κB/TNF pathway from pro-survival to pro-apoptosis.

It has been established that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) members promote survival by upregulating antiapoptotic genes and that genetic and pharmacological inhibition of NF-κB is required for tumor necrosis factor (TNF)-induced hepatocyte apoptosis. In this study, we demonstrate that this pro-survival pathway is switched to pro-apoptosis under pyruvate dehydrogenase kinase 4 (PDK4)-deficient conditions. PDK4-deficiency triggered hepatic apoptosis concomitantly with increased numbers of aberrant mitochondria, reactive oxygen species (ROS) production, sustained c-Jun N-terminal Kinase (JNK) activation, and reduction of glutathione (GSH). Interestingly, PDK4 retained p65 in cytoplasm via a direct protein-protein interaction. Disruption of PDK4-p65 association promoted p65 nuclear translocation. This, in turn, facilitated p65 binding to the TNF promoter to activate TNF-TNFR1 apoptotic pathway. Pdk4-/- livers were sensitized to Jo2 and D-(+)-Galactosamine /Lipopolysaccharide (GalN/LPS)-mediated apoptotic injury which was prevented by the inhibition of p65 or TNFR1. The pro-survival activity of TNF was shifted, which was switched to a pro-apoptotic activity in Pdk4-/- hepatocytes as a result of impaired activation of pro-survival NF-κB targets. Conclusion: PDK4 is indispensable to dictate the fate of TNF/NF-κB-mediated hepatocyte apoptosis. (Hepatology 2018).

DNA-mediated dimerization on a compact sequence signature controls enhancer engagement and regulation by FOXA1.

FOXA1 is a transcription factor capable to bind silenced chromatin to direct context-dependent cell fate conversion. Here, we demonstrate that a compact palindromic DNA element (termed ‘DIV’ for its diverging half-sites) induces the homodimerization of FOXA1 with strongly positive cooperativity. Alternative structural models are consistent with either an indirect DNA-mediated cooperativity or a direct protein-protein interaction. The cooperative homodimer formation is strictly constrained by precise half-site spacing. Re-analysis of chromatin immunoprecipitation sequencing data indicates that the DIV is effectively targeted by FOXA1 in the context of chromatin. Reporter assays show that FOXA1-dependent transcriptional activity declines when homodimeric binding is disrupted. In response to phosphatidylinositol-3 kinase inhibition DIV sites pre-bound by FOXA1 such as at the PVT1/MYC locus exhibit a strong increase in accessibility suggesting a role of the DIV configuration in the chromatin closed-open dynamics. Moreover, several disease-associated single nucleotide polymorphisms map to DIV elements and show allelic differences in FOXA1 homodimerization, reporter gene expression and are annotated as quantitative trait loci. This includes the rs541455835 variant at the MAPT locus encoding the Tau protein associated with Parkinson's disease. Collectively, the DIV guides chromatin engagement and regulation by FOXA1 and its perturbation could be linked to disease etiologies.

Type II toxin/antitoxin system ParESO /CopASO stabilizes prophage CP4So in Shewanella oneidensis.

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.

Limit-cycle oscillatory coexpression of cross-inhibitory transcription factors: a model mechanism for lineage promiscuity.

Lineage switches are genetic regulatory motifs that govern and maintain the commitment of a developing cell to a particular cell fate. A canonical example of a lineage switch is the pair of transcription factors PU.1 and GATA-1, of which the former is affiliated with the myeloid and the latter with the erythroid lineage within the haematopoietic system. On a molecular level, PU.1 and GATA-1 positively regulate themselves and antagonize each other via direct protein-protein interactions. Here we use mathematical modelling to identify a novel type of dynamic behaviour that can be supported by such a regulatory architecture. Guided by the specifics of the PU.1-GATA-1 interaction, we formulate, using the law of mass action, a system of differential equations for the key molecular concentrations. After a series of systematic approximations, the system is reduced to a simpler one, which is tractable to phase-plane and linearization methods. The reduced system formally resembles, and generalizes, a well-known model for competitive species from mathematical ecology. However, in addition to the qualitative regimes exhibited by a pair of competitive species (exclusivity, bistable exclusivity, stable-node coexpression) it also allows for oscillatory limit-cycle coexpression. A key outcome of the model is that, in the context of cell-fate choice, such oscillations could be harnessed by a differentiating cell to prime alternately for opposite outcomes; a bifurcation-theory approach is adopted to characterize this possibility.

The Nitrogen Regulatory PII Protein (GlnB) and N-Acetylglucosamine 6-Phosphate Epimerase (NanE) Allosterically Activate Glucosamine 6-Phosphate Deaminase (NagB) in Escherichia coli.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

Ring Finger Protein 207 Degrades T613M Kv11.1 Channel

Introduction: Human ether-à-go-go related gene (hERG, Kv11.1) channel underlies cardiac rapidly activating delayed rectifier K+ current (IKr); hERG mutations contribute to Long QT Syndrome (LQTS). Additionally, reduced repolarization reserve can arise from not only ion channel mutations, but also mutations in ion channel interacting proteins. RING Finger Protein 207 (RNF207) has been identified as a potential modifier for Kv11.1 function, cardiac excitation, and QT interval. Recent studies suggested a link between hERG and RNF207 mutations in LQTS onset and severity, specifically T613M hERG (hERGT613M) and RNF207 frameshift mutation (RNF207G603fs). However, exact molecular mechanisms remain unknown. We hypothesize that RNF207G603fs fails to degrade hERGT613M, allowing a higher population of membrane-bound hERGT613M mutant subunits, thus decreasing functional IKr. Methods and Results: We confirmed ubiquitinylation activity in wild-type RNF207 (RNF207WT). Confocal and super-resolution microscopy in murine and rabbit ventricular cardiomyocytes confirmed co-localization of hERG with RNF207. Functional analyses revealed that the trafficking-defective hERGT613M failed to produce currents. Wild-type hERG (hERGWT) subunits only partially rescued hERGT613M with reduced IKr and faster activation and inactivation kinetics. RNF207WT co-expression with hERGWT and hERGT613M restored IKr, whereas RNF207G603fs failed to rescue IKr. Co-immunoprecipitation of hERGWT pulled down RNF207WT, but not RNF207G603fs. Further analysis using cytoplasmic N- and C-terminal hERGWT deletions (hERGWT-ΔN, hERGWT-ΔC) revealed that RNF207WT pulled down hERGWT-ΔN, but not hERGWT-ΔC. Additionally, time-dependent degradation assays demonstrated increased hERGT613M degradation by RNF207WT, but not RNF207G603fs, via proteasomal degradation. We further tested changes to RNF207 structure using Rosetta molecular modeling software. Conclusion: RNF207WT functions as an E3 ubiquitin ligase for hERG channels. RNF207G603fs, however, failed to degrade hERGT613M and rescue IKr density, suggesting that direct protein-protein interaction is necessary for observed rescue of current. Our data suggest that normal RNF207 function is critical for hERG quality control and, consequently, cardiac repolarization.

Protein Interactomics by Two-Hybrid Methods.

Comprehensive identification of direct, physical interactions between biological macromolecules, such as protein-protein, protein-DNA, and protein-RNA interactions, is critical for our understanding of the function of gene products as well as the global organization and interworkings of various molecular machines within the cell. The accurate and comprehensive detection of direct interactions, however, remains a huge challenge due to the inherent structural complexity arising from various post-transcriptional and translational modifications coupled with huge heterogeneity in concentration, affinity, and subcellular location differences existing for any interacting molecules. This has created a need for developing multiple orthogonal and complementary assays for detecting various types of biological interactions. In this introduction, we discuss the methods developed for measuring different types of molecular interactions with an emphasis on direct protein-protein interactions, critical issues for generating high-quality interactome datasets, and the insights into biological networks and human diseases that current interaction mapping efforts provide. Further, we will discuss what future might lie ahead for the continued evolution of two-hybrid methods and the role of interactomics for expanding the advancement of biomedical science.

Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy.

Nasopharyngeal carcinoma (NPC) has a particularly high prevalence in southern China, southeastern Asia and northern Africa. Radiation resistance remains a serious obstacle to successful treatment in NPC. This study aimed to explore the metabolic feature of radiation-resistant NPC cells and identify new molecular-targeted agents to improve the therapeutic effects of radiotherapy in NPC. Methods: Radiation-responsive and radiation-resistant NPC cells were used as the model system in vitro and in vivo. Metabolomics approach was used to illustrate the global metabolic changes. 13C isotopomer tracing experiment and Seahorse XF analysis were undertaken to determine the activity of fatty acid oxidation (FAO). qRT-PCR was performed to evaluate the expression of essential FAO genes including CPT1A. NPC tumor tissue microarray was used to investigate the prognostic role of CPT1A. Either RNA interference or pharmacological blockade by Etomoxir were used to inhibit CPT1A. Radiation resistance was evaluated by colony formation assay. Mitochondrial membrane potential, apoptosis and neutral lipid content were measured by flow cytometry analysis using JC-1, Annexin V and LipidTOX Red probe respectively. Molecular markers of mitochondrial apoptosis were detected by western blot. Xenografts were treated with Etomoxir, radiation, or a combination of Etomoxir and radiation. Mitochondrial apoptosis and lipid droplets content of tumor tissues were detected by cleaved caspase 9 and Oil Red O staining respectively. Liquid chromatography coupled with tandem mass spectrometry approach was used to identify CPT1A-binding proteins. The interaction of CPT1A and Rab14 were detected by immunoprecipitation, immunofluorescence and in situ proximity ligation analysis. Fragment docking and direct coupling combined computational protein-protein interaction prediction method were used to predict the binding interface. Fatty acid trafficking was measured by pulse-chase assay using BODIPY C16 and MitoTracker Red probe. Results: FAO was active in radiation-resistant NPC cells, and the rate-limiting enzyme of FAO, carnitine palmitoyl transferase 1 A (CPT1A), was consistently up-regulated in these cells. The protein level of CPT1A was significantly associated with poor overall survival of NPC patients following radiotherapy. Inhibition of CPT1A re-sensitized NPC cells to radiation therapy by activating mitochondrial apoptosis both in vitro and in vivo. In addition, we identified Rab14 as a novel CPT1A binding protein. The CPT1A-Rab14 interaction facilitated fatty acid trafficking from lipid droplets to mitochondria, which decreased radiation-induced lipid accumulation and maximized ATP production. Knockdown of Rab14 attenuated CPT1A-mediated fatty acid trafficking and radiation resistance. Conclusion: An active FAO is a vital signature of NPC radiation resistance. Targeting CPT1A could be a beneficial regimen to improve the therapeutic effects of radiotherapy in NPC patients. Importantly, the CPT1A-Rab14 interaction plays roles in CPT1A-mediated radiation resistance by facilitating fatty acid trafficking. This interaction could be an attractive interface for the discovery of novel CPT1A inhibitors.

High Affinity Binding of Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) to Lu/BCAM Adhesion Glycoprotein

The protein toxin Cytotoxic Necrotizing Factor 1 (CNF1) is a major virulence factor of pathogenic Escherichia coli strains. It belongs to a family of single chain AB-toxins, which enter mammalian cells by receptor-mediated endocytosis. Recently, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as a cellular receptor for CNF1. Here, we identified the Ig-like domain 2 of Lu/BCAM as main interaction site of the toxin by direct protein-protein interaction and competition studies. Using surface plasmon resonance, we showed a high affinity CNF-Lu/BCAM interaction with a KD of 2.8 nM. Furthermore, we performed small-angle X-ray scattering to define the molecular envelope of the Lu/BCAM-CNF1 complex, suggesting a 6:1 ratio of Lu/BCAM to CNF1 in the receptor-toxin complex. This study leads to a deeper understanding of the interaction between CNF1 and Lu/BCAM, and presents novel opportunities for the development of future anti-toxin strategies.

New insights into the mechanisms of phytochrome-cryptochrome coaction.

Contents Summary 547 I. Introduction 547 II. Phytochromes mediate light-induced transcription of BICs to inactivate cryptochromes 548 III. PPKs phosphorylate light-signaling proteins and histones to affect plant development 548 IV. Prospect 550 Acknowledgements 550 References 550 SUMMARY: Plants perceive and respond to light signals by multiple sensory photoreceptors, including phytochromes and cryptochromes, which absorb different wavelengths of light to regulate genome expression and plant development. Photophysiological analyses have long revealed the coordinated actions of different photoreceptors, a phenomenon referred to as the photoreceptor coaction. The mechanistic explanations of photoreceptor coactions are not fully understood. The function of direct protein-protein interaction of phytochromes and cryptochromes and common signaling molecules of these photoreceptors, such as SPA1/COP1 E3 ubiquitin ligase complex and bHLH transcription factors PIFs, would partially explain phytochrome-cryptochrome coactions. In addition, newly discovered proteins that block cryptochrome photodimerization or catalyze cryptochrome phosphorylation may also participate in the phytochrome and cryptochrome coaction. This Tansley insight, which is not intended to make a comprehensive review of the studies of photoreceptor coactions, attempts to highlight those recent findings and their possible roles in the photoreceptor coaction.