Direct Physical Interaction Research Articles

Association of functional variants and protein-to-protein physical interactions of human MutY homolog linked with familial adenomatous polyposis and colorectal cancer syndrome.

The human gene MUTYH codes for a DNA glycosylase involved in the repair of oxidative DNA damage. Faulty MUTYH protein activity causes the accumulation of G→T transversions due to unrepaired 8-oxoG:A mismatches. MUTYH germ-line mutations in humans are linked with a recessive form of Familial Adenomatous Polyposis (FAP) and colorectal cancer predisposition. We studied the repair capacity of variants identified in MUTYH-associated polyposis (MAP) patients. MAP is inherited in an autosomal recessive type due to mutations in MUTYH (Y165C, G382D, P54S, A22V, Q63R, G45D, S136P and N43S), indicating that both copies of the gene become inactivated. However, the parents of an individual with an autosomal recessive condition may serve as carriers, each harboring one copy of the mutated gene without showing signs or symptoms of MAP. Six protein partners have been associated with MUTYH, four via direct physical interactions, namely, hMSH6, hPCNA, hRPA1, and hAPEX1. We examined, for the first time, specific interactions of these protein partners with MAP-associated MUTYH mutants using molecular dynamics simulations. The approach provided tools for exploration of the conformational energy landscape accessible to protein partners. The investigation also determined the impact before and after energy minimization of protein-protein interactions and binding affinities of MUTYH wild type and mutant forms, as well as the interactions with other proteins. Taken together, this study provided new insights into the role of MUTYH and its interacting proteins in MAP.

Direct physical interaction of active Ras with mSIN1 regulates mTORC2 signaling

BackgroundThe mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking.MethodsProtein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion.ResultsHere, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling.ConclusionsOur study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

DUF3669, a \u201cdomain of unknown function\u201d within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression

BackgroundZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the “domain of unknown function 3669” (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson’s disease.ResultsHere we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669’s impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved “MLE” sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein.ConclusionDUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

BBX4, a phyB-interacting and modulated regulator, directly interacts with PIF3 to fine tune red light-mediated photomorphogenesis

Phytochrome B (phyB) absorbs red light signals and subsequently initiates a set of molecular events in plant cells to promote photomorphogenesis. Here we show that phyB directly interacts with B-BOX CONTAINING PROTEIN 4 (BBX4), a positive regulator of red light signaling, and positively controls its abundance in red light. BBX4 associates with PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and represses PIF3 transcriptional activation activity and PIF3-controlled gene expression. The degradation of BBX4 in darkness is dependent on CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and the 26S proteasome system. Collectively, BBX4 acts as a key component of the phyB-PIF3-mediated signaling module and fine tunes the red light action. phyB promotes the accumulation of BBX4, which in turn serves to repress PIF3 action through direct physical interaction to promote photomorphogenic development in red light.

Inhibiting Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Induces Selective Leukemia Cell Death

Targeting cell metabolism has emerged as a viable treatment strategy in acute myeloid leukemia (AML), a malignant hematological disease characterized by poor patient outcomes and limited chemotherapeutic options. Compared to the normal hematopoietic population, leukemia cells exhibit an altered phenotype characterized by increased mitochondrial mass as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. Mitochondrial FAO is a four-reaction process that catabolizes fatty acids to acetyl-CoA, generating reductive equivalents for the electron transport chain (ETC) and anaplerotic intermediates for the TCA cycle. Clinically approved FAO inhibitors, such as trimetazidine and ranolazine, are tissue-specific, targeting FAO in some tissues but affecting additional pathways in others. To better understand the potential clinical utility of targeting FAO, we systematically tested a panel of clinical and pre-clinical FAO inhibitors, reasoning that the most potent FAO inhibitor would lead to a novel anti-AML target. Avocadyne was the most potent anti-AML compound, inducing leukemic cell death (EC50: 2.5 µM) and suppressing clonogenic growth of primary samples, while sparing normal hematopoietic cells. Further, avocadyne (100mg/kg twice weekly for 5 weeks) reduced patient-derived AML cell engraftment in the bone marrow of immune deficient mice. As a component of avocatin-B, a mixture of two fatty alcohols previously determined to accumulate in the mitochondria, we confirmed that avocadyne inhibited long chain FAO using radiolabeled studies and high resolution respirometry. To identify a molecular target, avocadyne treated cells were immunoprecipitated with antibodies against each intramitochondrial enzyme involved in long chain FAO (e.g., very long acyl-CoA dehydrogenase (VLCAD; step 1) and the alpha and beta subunits of the mitochondrial trifunctional protein: HADHA, HADHB; steps 2-4). Immunoblotting and LC/MS analysis confirmed that avocadyne co-eluted with VLCAD, but not with HADHA or HADHB, confirming a direct physical interaction between avocadyne and VLCAD. VLCAD introduces a double bond to a fully saturated long chain fatty acid and reduces electron transfer flavoprotein (ETF)-bound FAD, transferring these electrons to the ETC. Using fluorescence spectrophotometry and respirometry, avocadyne directly inhibited VLCAD activity, resulting in reduced ETF-supported respiration. The activity of MCAD, an acyl-CoA dehydrogenase catalyzing medium chain fats, was not affected, suggesting avocadyne inhibits long chain FAO exclusively. Further, profiling of acyl-carnitines following avocadyne treatment also showed a pattern characteristic of long chain FAO inhibition at VLCAD. To further understand how VLCAD modulated avocadyne sensitivity, avocadyne-resistant and VLCAD knockdown cells were generated. Lentiviral knockdown of VLCAD sensitized leukemic cells to avocadyne-induced FAO inhibition and death. In contrast, cells resistant to avocadyne had increased protein expression of VLCAD but no change in other long chain FAO enzymes. With increased VLCAD-supported ETF respiration, higher concentrations of avocadyne were required to induce cell death, compared to the parental line. These results show that VLCAD expression modulated leukemic sensitivity to avocadyne. Inhibiting FAO at VLCAD triggered an adaptive metabolic switch towards glycolysis, characterized by increased extracellular acidification. This compensatory increase in glycolysis was ultimately insufficient to prevent the depletion of TCA metabolites and ATP, leading to leukemic death. In contrast, following avocadyne treatment, normal umbilical cord blood-derived mononuclear cells increased glycolytic as well as pyruvate dehydrogenase activity and had no decrease in ATP levels, cell viability, or clonogenic growth. Together, these results highlight, for the first time, VLCAD as a novel anti-AML target and further suggest the clinical utility of FAO inhibition as a potential anti-AML strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Phylogenetic correlations can suffice to infer protein partners from sequences.

Determining which proteins interact together is crucial to a systems-level understanding of the cell. Recently, algorithms based on Direct Coupling Analysis (DCA) pairwise maximum-entropy models have allowed to identify interaction partners among paralogous proteins from sequence data. This success of DCA at predicting protein-protein interactions could be mainly based on its known ability to identify pairs of residues that are in contact in the three-dimensional structure of protein complexes and that coevolve to remain physicochemically complementary. However, interacting proteins possess similar evolutionary histories. What is the role of purely phylogenetic correlations in the performance of DCA-based methods to infer interaction partners? To address this question, we employ controlled synthetic data that only involve phylogeny and no interactions or contacts. We find that DCA accurately identifies the pairs of synthetic sequences that share evolutionary history. While phylogenetic correlations confound the identification of contacting residues by DCA, they are thus useful to predict interacting partners among paralogs. We find that DCA performs as well as phylogenetic methods to this end, and slightly better than them with large and accurate training sets. Employing DCA or phylogenetic methods within an Iterative Pairing Algorithm (IPA) allows to predict pairs of evolutionary partners without a training set. We further demonstrate the ability of these various methods to correctly predict pairings among real paralogous proteins with genome proximity but no known direct physical interaction, illustrating the importance of phylogenetic correlations in natural data. However, for physically interacting and strongly coevolving proteins, DCA and mutual information outperform phylogenetic methods. We finally discuss how to distinguish physically interacting proteins from proteins that only share a common evolutionary history.

Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease.

Misfolding, aggregation, and aberrant accumulation of proteins are central components in the progression of neurodegenerative disease. Cellular molecular chaperone systems modulate proteostasis, and, therefore, are primed to influence aberrant protein-induced neurotoxicity and disease progression. Molecular chaperones have a wide range of functions from facilitating proper nascent folding and refolding to degradation or sequestration of misfolded substrates. In disease states, molecular chaperones can display protective or aberrant effects, including the promotion and stabilization of toxic protein aggregates. This seems to be dependent on the aggregating protein and discrete chaperone interaction. Small heat shock proteins (sHsps) are a class of molecular chaperones that typically associate early with misfolded proteins. These interactions hold proteins in a reversible state that helps facilitate refolding or degradation by other chaperones and co-factors. These sHsp interactions require dynamic oligomerization state changes in response to diverse cellular triggers and, unlike later steps in the chaperone cascade of events, are ATP-independent. Here, we review evidence for modulation of neurodegenerative disease-relevant protein aggregation by sHsps. This includes data supporting direct physical interactions and potential roles of sHsps in the stewardship of pathological protein aggregates in brain. A greater understanding of the mechanisms of sHsp chaperone activity may help in the development of novel therapeutic strategies to modulate the aggregation of pathological, amyloidogenic proteins. sHsps-targeting strategies including modulators of expression or post-translational modification of endogenous sHsps, small molecules targeted to sHsp domains, and delivery of engineered molecular chaperones, are also discussed.

Decrypting noncoding RNA interactions, structures, and functional networks.

The world of noncoding RNAs (ncRNAs) is composed of an enormous and growing number of transcripts, ranging in length from tens of bases to tens of kilobases, involved in all biological processes and altered in expression and/or function in many types of human disorders. The premise of this review is the concept that ncRNAs, like many large proteins, have a multidomain architecture that organizes them spatially and functionally. As ncRNAs are beginning to be imprecisely classified into functional families, we review here how their structural properties might inform their functions with focus on structural architecture–function relationships. We will describe the properties of “interactor elements” (IEs) involved in direct physical interaction with nucleic acids, proteins, or lipids and of “structural elements” (SEs) directing their wiring within the “ncRNA interactor networks” through the emergence of secondary and/or tertiary structures. We suggest that spectrums of “letters” (ncRNA elements) are assembled into “words” (ncRNA domains) that are further organized into “phrases” (complete ncRNA structures) with functional meaning (signaling output) through complex “sentences” (the ncRNA interactor networks). This semiotic analogy can guide the exploitation of ncRNAs as new therapeutic targets through the development of IE-blockers and/or SE-lockers that will change the interactor partners’ spectrum of proteins, RNAs, DNAs, or lipids and consequently influence disease phenotypes.

Reciprocal signaling and direct physical interactions between fibroblasts and breast cancer cells in a 3D environment.

Tumorigenic cells undergo cell aggregation and aggregate coalescence in a 3D Matrigel environment. Here, we expanded this 3D platform to assess the interactions of normal human dermal fibroblasts (NHDFs) and human primary mammary fibroblasts (HPMFs) with breast cancer-derived, tumorigenic cells (MDA-MB-231). Medium conditioned by MDA-MB-231 cells activates both types of fibroblasts, imbuing them with the capacity to accelerate the rate of aggregation and coalescence of MDA-MB-231 cells more than four fold. Acceleration is achieved 1) by direct physical interactions with MDA-MB-231 cells, in which activated fibroblasts penetrate the MDA-MB-231/Matrigel 3D environment and function as supporting scaffolds for MDA-MB-231 aggregation and coalescence, and 2) through the release of soluble accelerating factors, including matrix metalloproteinase (MMPs) and, in the case of activated NHDFs, SDF-1α/CXCL12. Fibroblast activation includes changes in morphology, motility, and gene expression. Podoplanin (PDPN) and fibroblast activation protein (FAP) are upregulated by more than nine-fold in activated NHDFs while activated HPMFs upregulate FAP, vimentin, desmin, platelet derived growth factor receptor A and S100A4. Overexpression of PDPN, but not FAP, in NHDF cells in the absence of MDA-MB-231-conditioned medium, activates NHDFs. These results reveal that complex reciprocal signaling between fibroblasts and cancer cells, coupled with their physical interactions, occurs in a highly coordinated fashion that orchestrates aggregation and coalescence, behaviors specific to cancer cells in a 3D environment. These in vitro interactions may reflect events involved in early tumorigenesis, particularly in cases of field cancerization, and may represent a new mechanism whereby cancer-associated fibroblasts (CAFs) promote tumor growth.

Regulation by competition: a hidden layer of gene regulatory network

BackgroundMolecular competition brings about trade‐offs of shared limited resources among the cellular components, and thus introduces a hidden layer of regulatory mechanism by connecting components even without direct physical interactions. Several molecular competition scenarios have been observed recently, but there is still a lack of systematic quantitative understanding to reveal the essence of molecular competition.MethodsHere, by abstracting the analogous competition mechanism behind diverse molecular systems, we built a unified coarse‐grained competition motif model to systematically integrate experimental evidences in these processes and analyzed general properties shared behind them from steady‐state behavior to dynamic responses.ResultsWe could predict in what molecular environments competition would reveal threshold behavior or display a negative linear dependence. We quantified how competition can shape regulator‐target dose‐response curve, modulate dynamic response speed, control target expression noise, and introduce correlated fluctuations between targets.ConclusionsThis work uncovered the complexity and generality of molecular competition effect as a hidden layer of gene regulatory network, and therefore provided a unified insight and a theoretical framework to understand and employ competition in both natural and synthetic systems.

Molecular basis of allosteric Orai1 channel activation by STIM1.

Store-operated Ca2+ entry through Orai1 channels is a primary mechanism for Ca2+ entry in many cells and mediates numerous cellular effector functions ranging from gene transcription to exocytosis. Orai1 channels are amongst the most Ca2+ -selective channels known and are activated by direct physical interactions with the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) in response to store depletion triggered by stimulation of a variety of cell surface G-protein coupled and tyrosine kinase receptors. Work in the last decade has revealed that the Orai1 gating process is highly cooperative and strongly allosteric, likely driven by a wave of interdependent conformational changes throughout the protein originating in the peripheral C-terminal ligand binding site and culminating in pore opening. In this review, we survey the structural and molecular features in Orai1 that contribute to channel gating and consider how they give rise to the unique biophysical fingerprint of Orai1 currents.

Myosin Va and spermine synthase: partners in exosome transport.

A recent paper in Bioscience Reports (BSR20182189) describes the discovery of an interaction between the motor protein myosin Va and the metabolic enzyme spermine synthase. Myosin Va is a molecular motor which plays a key role in vesicle transport. Mutations in the gene which encodes this protein are associated with Griscelli syndrome type 1 and the ‘dilute’ phenotype in animals. Spermine synthase catalyzes the conversion of spermidine to spermine. This largely cytoplasmic enzyme can also be localized to the soluble fraction in exosomes. Mutations in the spermine synthase gene are associated with Snyder Robinson mental retardation syndrome. The interaction between the two proteins was detected using the yeast two hybrid method and verified by microscale thermophoresis of recombinant proteins. Knockdown of the MYO5A gene reduced the expression of mRNA coding for spermine synthase. The amount of this transcript was also reduced in cells derived from a patient with Griscelli syndrome type 1. This suggests that, in addition to a direct physical interaction between the two proteins, myosin Va also modulates the transcription of the spermine synthase gene. The mechanism for this modulation is currently unknown. These findings have implications for Griscelli syndrome type 1 and Snyder Robinson mental retardation syndrome. They also suggest that interactions between myosin Va and soluble exosome proteins such as spermine synthase may be important in the mechanism of exosome transport.

New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and its mitotic kinase Aurora A

The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the Km value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177–197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min−1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.

Isolation of a Novel Complex Between Human NER Proteins XPC and XPA

Nucleotide excision repair (NER) is the primary mechanism to remove large covalent DNA lesions resulting from exposure to a wide variety of genotoxic agents. The human NER mechanism is dependent on the coordinated activity of over thirty proteins that build a series of protein machines to repair the damaged DNA. Two proteins, XPC and XPA, play critical roles in NER despite lacking any enzymatic activity. XPC serves as a generic sensor of damaged DNA, marking a site for repair and initiating assembly of the NER machinery. XPA is generally believed to act as a molecular scaffold upon which the NER machines are built. Previous studies have suggested a weak but direct physical interaction between XPC and XPA, and genetic evidence suggests this interaction may aid in recruitment of XPA to potential damage sites in cells. In this study, we identified a novel protein complex between human XPA and the globular transglutaminase-like domain of XPC (XPC-TGD). We employed a chemical cross-linking strategy to isolate protein complexes formed from highly purified recombinant proteins. Complexes were purified by SDS-PAGE and subjected to trypsin proteolysis. MALDI-TOF mass spectrometry analysis identified polypeptide fragments from both the XPA and XPC proteins in tryptic digests of isolated complex samples. These results suggest the XPA may interact with XPC by binding to the XPC-TGD during assembly of the NER machinery. Support or Funding Information Supported by The University of Virginia's College at Wise Fellowship in Natural Sciences (FINS) Undergraduate Research Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Source-Sink Regulation Is Mediated by Interaction of an FT Homolog with a SWEET Protein in Potato

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch maybe mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.

Functional analysis of the Chloroplast GrpE (CGE) proteins from Arabidopsis thaliana

The function of proteins depends on specific partners that regulate protein folding, degradation and protein-protein interactions, such partners are the chaperones and cochaperones. In chloroplasts, proteins belonging to several families of chaperones have been identified: chaperonins (Cpn60s), Hsp90s (Hsp90-5/Hsp90C), Hsp100s (Hsp93/ClpC) and Hsp70s (cpHsc70s). Several lines of evidence have demonstrated that cpHsc70 chaperones are involved in molecular processes like protein import, protein folding and oligomer formation that impact important physiological aspects in plants such as thermotolerance and thylakoid biogenesis. Despite the vast amount of data existing around the function of cpHcp70s chaperones, very little attention has been paid to the roles of DnaJ and GrpE cochaperones in the chloroplast. In this study, we performed a phylogenetic analysis of the chloroplastic GrpE (CGE) proteins from 71 species. Based on their phylogenetic relationships and on a motif enrichment analysis, we propose a classification system for land plants’ CGEs, which include two independent groups with specific primary structure traits. Furthermore, using in vivo assays we determined that the two CGEs from A. thaliana (AtCGEs) complement the mutant phenotype displayed by a knockout E. coli strain defective in the bacterial grpE gene. Moreover, we determined in planta that the two AtCGEs are bona fide chloroplastic proteins, which form the essential homodimers needed to establish direct physical interactions with the cpHsc70-1 chaperone. Finally, we found evidence suggesting that AtCGE1 is involved in specific physiological phenomena in A. thaliana, such as the chloroplastic response to heat stress, and the correct oligomerization of the photosynthesis-related LHCII complex.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400–403), which is located within the CAD/SOAR domain. We determined this segment’s specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation.

DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11-RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation-dependent mode through NBS1 and a phosphorylation-independent mode without NBS1. In support, we show that limited DNA end resection occurs invivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11-RAD50 nuclease to S-G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.

Abstract B159: Investigating the neuroimmune interaction between nociceptive neurons and dendritic cells

Abstract Nociceptive somatosensory neurons (nociceptors) have recently been established as controllers of immune responses in the context of infections, allergic airway inflammation, and imiquimod (IMQ)-induced psoriasiform skin inflammation. In most of these models, myeloid cells including dendritic cells (DCs) were shown to be a target of the actions of nociceptors. DCs form a bridge between the innate and adaptive immune system and it is mostly through their actions that the adaptive immune system gets activated. Notably, DCs were previously found to engage in a direct physical interaction with nociceptors as well as to be affected by soluble neuropeptides produced by the latter. Conflicting results have, however, been reported with regards to the function and the requirement of neuropeptides for the nociceptor:DC interaction under in vivo settings, suggesting a degree of context-dependency, while the requirement for physical interaction of the two cell types has not been tested. Inherently, in vivo approaches are not readily amenable for dissection of the molecular underpinnings of the interaction, and involvement of other, accessory cells can be difficult to exclude. Consequently, we have established an in-vitro nociceptor:DC co-culture system, which, compared to in vivo settings, allows us to dissect nociceptor:DC interactions in a less complex, reductionist setting.Using the in vitro co-culture setup, we show that dendritic cells and nociceptors engage in tight physical interactions, as well as that nociceptors produce chemo-attractant signals, which act directly on DCs. Further, we demonstrate that nociceptors enhance production of IL-6 and IL12-p40 cytokines by dendritic cells in response to IMQ, a TLR7 agonist. Strikingly, this effect was only apparent when DCs and nociceptors were allowed direct contact, suggesting a contact or proximity-based mechanism of interaction. In addition to IMQ, we identify other pathogen-associated molecular pattern (PAMP) molecules, which elicit a cytokine response that can be enhanced in the presence of nociceptors. Next, using live cell imaging, we showed that, as described previously, treatment of nociceptors with capsaicin, a TRPV-1 channel agonist, results in calcium flux and membrane depolarization of the neuronal cells. Strikingly, similar changes can be observed in DCs interacting with the neurons, while DCs alone do not exhibit any such behavior when treated with capsaicin. To further substantiate our findings, we utilized channelrhodopsin-expressing neurons, which can be activated by blue light without any other perturbations of the system. Importantly, light-induced activation of such neurons was sufficient to drive a calcium flux in the interacting DCs.Finally, to gain an unbiased view of the effects that nociceptors have on DCs, we performed RNA sequencing of DCs after co-culture. Strikingly, we observed profound changes in gene expression across DC subsets when DCs were cultured with neurons without any further stimulation as well as when they were treated with IMQ in the co-culture.In summary, we have developed an in vitro system that allows us to investigate the interactions between nociceptors and DCs. Using this system, we have confirmed that nociceptors directly communicate with DCs, we addressed the requirement for physical interactions of the two cell types and we have begun to shed light on the molecular underpinnings of these interactions. Citation Format: Pavel Hanc, Siyi Huang, Ulrich H. von Andrian. Investigating the neuroimmune interaction between nociceptive neurons and dendritic cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B159.

Abstract TP322: Viral Mediated Gene Delivery of TMBIM6 Protects the Neonatal Brain

Introduction: A wide variety of conditions may contribute to oxidative protein folding processes in the endoplasmic reticulum (ER) which cause ER stress and play a major role in the pathogenesis of neonatal hypoxic ischemic (HI) injury. ER stress triggers the unfolded protein response (UPR) resulting in the activation of pro-apoptotic signaling causing cells to undergo apoptosis. Here were explored the role of Bax Inhibitor-1 (BI-1) protein, an evolutionary protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, as a novel modulator of ER stress induced apoptosis after HI brain injury in a neonatal rat pup. BI-1 may modulate the UPR response via a direct physical interaction with the stress sensor receptor, IRE1α, and thus inhibiting it’s pro-apoptotic signaling. Hypothesis: Overexpression of BI-1, using viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, will protect the neonatal brain from HI by inhibiting ER stress induced apoptosis. Methods: Ten-day old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). To determine BI-1 confers protection via inhibition of IRE1α, we administered BI-1 siRNA, to silence BI-1, and an IRE1α CRISPR activation plasmid (AP), to counteract BI-1’s effects, at 48h pre-HI. Percent infarct area, immunofluorescent staining and western blot were performed at 72h post HI. Results: Overexpression of BI-1 showed to significantly reduce percent infarcted area, attenuated neuronal degeneration, reduced ER stress induced neuronal apoptosis and improved long-term neurological outcomes. Either silencing BI-1 or over activating IRE1α with a CRISPR AP significantly reversed BI-1’s protective effects. Conclusions: Overexpression of BI-1 attenuated the morphological and neurological consequences post neonatal HI via inhibition of the ER stress sensor, IRE1α. This study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and provided a basis for BI-1 as a potential therapeutic target.