P. gingivalis-Induced TLR2 Interactome Analysis Reveals Association with PARP9.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

Detecting Cardiovascular Protein-Protein Interactions by Proximity Proteomics.

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.

Identification of Full-Length Wild-Type and Mutant Huntingtin Interacting Proteins by Crosslinking Immunoprecipitation in Mice Brain Cortex.

BACKGROUND: Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein. OBJECTIVE: Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington's disease pathology. METHODS: Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2. CONCLUSION: Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington's disease pathology.

Introducing APOA1 as a key protein in COVID-19 infection: a bioinformatics approach.

AIM: Introducing possible diagnostic and therapeutic biomarker candidates via the identification of chief dysregulated proteins in COVID-19 patients is the aim of this study. BACKGROUND: Molecular studies, especially proteomics, can be considered as suitable approaches for discovering the hidden aspect of the disease. METHODS: Differentially expressed proteins (DEPs) of three patients with demonstrated severe condition (S-COVID-19) were compared to healthy cases by a proteomics study. Cytoscape software and STRING database were used to construct the protein-protein interaction (PPI) network. The central DEPs were identified through topological analysis of the network. ClueGO+CluePedia were applied to find the biological processes related to the central nodes. MCODE molecular complex detection (MCODE) was used to discover protein complexes. RESULTS: A total of 242 DEPs from among 256 query ones were included in the network. Centrality analysis of the network assigned 16 hub-bottlenecks, nine of which were presented in the highest-scored protein complex. Ten protein complexes were determined. APOA1 was identified as the protein complex seed, and APP, EGF, and C3 were the top hub-bottlenecks of the network. The results specify that up-regulation of C3 and down-regulation of APOA1 in urine play a role in the stiffness in respiration and, accordingly, the severity of COVID-19. Moreover, dysregulation of APP and APOA1 could both contribute to the possible adverse effects of COVID-19 on the nervous system. CONCLUSION: The introduced central proteins of the S-COVID-19 interaction network, particularly APOA1, can be considered as diagnostic and therapeutic targets related to the coronavirus disease after being approved with complementary studies.

Using the pathogen-host interactions database (PHI-base) to investigate plant pathogen genomes and genes implicated in virulence.

New pathogen-host interaction mechanisms can be revealed by integrating mutant phenotype data with genetic information. PHI-base is a multi-species manually curated database combining peer-reviewed published phenotype data from plant and animal pathogens and gene/protein information in a single database.

HEV-Capsid Protein Interacts With Cytochrome P4502C8 and Retinol-Binding Protein 4.

BACKGROUND: Hepatitis E virus (HEV) is a major causative agent of acute clinical hepatitis in adults throughout much of Asia, the Middle East, and Africa. The lack of an efficient cell culture system for HEV has greatly limited our understanding of the mechanisms of infection, replication, and pathogenicity of this virus. The yeast two-hybridization system is considered to be an efficient method for determining protein-protein interactions and screening interactive proteins associated with host cells. OBJECTIVES: In order to identify the host-cell proteins interacting with the HEV-capsid proteins, a fragment of the HEV-capsid protein p239 (amino acids 368-606) was used as bait; human liver cDNA library was used as a source of host-cell proteins, and the screening was performed using the CytoTrap yeast two-hybrid system. MATERIALS AND METHODS: The CytoTrap yeast two-hybrid system, which is also called Sos Recruitment System (SRS), was used to analyze the interaction of the p239 fragment with host-cell proteins. RESULTS: We isolated 2 proteins, cytochrome P4502C8 (CYP4502C8) and retinol-binding protein 4 (RBP4) after 2 rounds of screening. Co-immunoprecipitation assays showed that both the proteins could bind in vitro to the HEV virion in HepG2 cells. CONCLUSIONS: CYP4502C8 and RBP4 screened from liver cDNA library using the CytoTrap yeast two-hybrid system interact with HEV capsid in vitro.

Ubiquitylation of Fe65 adaptor protein by neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) via the WW domain interaction with Fe65

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (72PPLP75) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (72PPLP75 was changed to 72APLA75) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.

Langerhans cell protein 1 (LCP1) binds to PNUTS in the nucleus: implications for this complex in transcriptional regulation

Protein phosphatase-1 (PP1) nuclear targeting subunit (PNUTS), also called PP1R10, p99, or CAT 53 was originally isolated as a mammalian nuclear PP1-binding protein. In this study, we performed yeast two-hybrid screens to identify PNUTS-interacting proteins. Here, we report that LCP1 (epidermal Langerhans cell protein 1), a novel member of the HMG-box protein family, binds tightly to PNUTS. Co-immunoprecipitation of deletion constructs revealed that the C-terminus of LCP1 is sufficient for the interaction with an N-terminal region of PNUTS that is distinct from its PP1-binding domain. Furthermore, immunofluorescence studies showed that a subpopulation of LCP1 co-localizes with PNUTS in nuclear speckles. Importantly, we found that the N-terminus of LCP1 has a strong trans-activation activity in a GAL4-based heterologous transcription assay. The transcriptional activity of LCP1 is markedly suppressed by its interaction with PNUTS, in a PP1-independent manner. These findings suggest that the coordinated spatial and temporal regulation of LCP1 and PNUTS may be a novel mechanism to control the expression of genes that are critical for certain physiological and pathological processes.

Regulation of Wnt signaling by protein-protein interaction and post-translational modifications

The Wnt signaling pathway is conserved in various species from worms to mammals, and plays important roles in cellular proliferation, differentiation, and migration. Wnt stabilizes cytoplasmic beta-catenin and then the accumulated beta-catenin is translocated into the nucleus, where it activates the transcriptional factor T-cell factor (Tcf)/lymphoid enhancer factor (Lef), and thereby stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Tight regulation of this response involves post-translational modifications of the components of the Wnt signaling pathway. Phosphorylation, ubiquitination, and sumoylation have been shown to affect the half-life of beta-catenin and the transcriptional activity of Tcf/Lef. The precise spatio-temporal patterns of these multiple modifications determine the driving force of various cellular responses.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60v-src in v-src-transformed Rat-1 fibroblasts

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kappa Da in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60v-src in vivo and CPTP1 also can associate with p60v-src in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60v-src was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60v-src in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kappa Da protein which has a possibility to be tyrosine- phosphorylated by p60v-src in v-src-transformed Rat- 1 fibroblasts. These results suggest that HPTP1B- type PTPs may play an important role in p60src dependent signal pathway in eucaryotic cells.