Identification of protein complexes from co-immunoprecipitation data

Motivation: Advanced technologies are producing large-scale protein–protein interaction data at an ever increasing pace. A fundamental challenge in analyzing these data is the inference of protein machineries. Previous methods for detecting protein complexes have been mainly based on analyzing binary protein–protein interaction data, ignoring the more involved co-complex relations obtained from co-immunoprecipitation experiments

Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex

BACKGROUND: Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry. PRINCIPLE FINDINGS: The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues. CONCLUSION: Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders

The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGEhomology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5–6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Introduction: Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods: IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results: Circulating IC in the serum of RA patients and healthy controls contain fibrinogen beta and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogen. and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogen beta were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions: Citrullinated fibrinogen beta and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA-RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA

The ever-evolving concept of the gene: The use of RNA/Protein experimental techniques to understand genome functions

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as "junk" DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years

ADENYLYL CYCLASE TYPE 9: REGULATION AND CARDIAC FUNCTION

Abnormalities in cardiac stress signaling underlie a number of cardiovascular diseases (e.g. arrhythmias and heart failure). Cardiac stress signaling pathways normally integrate signals from the sympathetic nervous system to promote efficient contraction and relaxation under stress. Sympathetic control through β-adrenergic stimulation is propagated by adenylyl cyclase (AC). AC synthesizes cyclic AMP (cAMP), an important second messenger that initiates signaling pathways to modulate physiological and pathophysiological functions of the heart, including the activation of PKA and subsequent phosphorylation of ion channels, contractile machinery, and stress response proteins that enhance cardiac function. Alterations of cAMP signaling occur in the failing heart and contribute to impaired function. Of the AC isoforms present in adult cardiomyocytes (AC 4, 5, 6, and 9), AC9 is the most divergent in sequence and understudied. The work presented in this dissertation sought to evaluate the direct regulatory properties of AC9 and explores roles for AC9 in heart. To clarify conflicting reports for AC9 regulation, proposed regulators were systematically evaluated, including G-proteins, protein kinases, and forskolin utilizing in vitro and cell based assays. Overall, I conclude that most G-proteins or protein kinases do not directly regulate AC9, except Gαs, in vitro. Although AC9 is forskolin insensitive alone, weak activation by forskolin in the presence of Gαs is possible. AC9 shows significant homodimerization and modest heterodimerization with AC5/6, which may account for the conflicting reports surrounding the regulation of this AC isoform. viii To study the role of AC9 in heart, a mouse model of AC9 genetic deletion was utilized. Although deletion of AC9 reduces less than 3% of total AC activity in heart, Yotiao-associated AC activity is eliminated. AC9-/- mice exhibit no structural abnormalities but show a significant bradycardia and alterations in Doppler echocardiography indicative of grade 1 diastolic dysfunction with preserved ejection fraction. Identification of novel AC9 binding partners, including the small heat shock protein 20 (Hsp20) and Popeye domain containing (Popdc) proteins may contribute to the underlying mechanisms of AC9-/- phenotypes. Collectively, this work suggests that AC9 forms distinct macromolecular complexes that contribute to local cAMP pools important for driving physiological function of the heart

RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

Background: Due to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species. Results: Here we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins. Conclusions: The RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.Fil: Marmisollé, Facundo Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

The Arabidopsis Malectin-Like/LRR-RLK IOS1 is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity

Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR) and CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent beta-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hyper-susceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed up-regulation of the PTI-marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1), reduced callose deposition and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines over-expressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, co-immunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner, and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitin-mediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR- and CERK1-mediated signaling pathways that primes PTI activation

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways