An integer programming framework for inferring disease complexes from network data

MOTIVATION: Unraveling the molecular mechanisms that underlie disease calls for methods that go beyond the identification of single causal genes to inferring larger protein assemblies that take part in the disease process. RESULTS: Here, we develop an exact, integer-programming-based method for associating protein complexes with disease. Our approach scores proteins based on their proximity in a protein-protein interaction network to a prior set that is known to be relevant for the studied disease. These scores are combined with interaction information to infer densely interacting protein complexes that are potentially disease-associated. We show that our method outperforms previous ones and leads to predictions that are well supported by current experimental data and literature knowledge. AVAILABILITY AND IMPLEMENTATION: The datasets we used, the executables and the results are available at www.cs.tau.ac.il/roded/disease_complexes.zip. CONTACT: [email protected]

DULIP: A dual luminescence-based co-immunoprecipitation assay for interactome mapping in mammalian cells

Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening as well as for the validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems

Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.DFG [SFB740, 740/2-11, SFB618, 618/3-09, SFB/TRR43 A7]; BMBF(NGFN-Plus) [01GS08169-73, 01GS08150, 01GS08108]; HDSA Coalition for the Cure; EU (EuroSpin) [Health-F2-2009-241498, HEALTH-F2-2009-242167]; Helmholtz Association (MSBN, HelMA) [HA-215]; FCT [IF/00881/2013]info:eu-repo/semantics/publishedVersio

Efficacy of nadph-oxidase and rho-kinase pathways inhibition in a genetic in-vitro Parkinson's disease model

SummaryThe nerve terminal protein α-synuclein and its interacting protein synphilin-1 are major protein components of Lewy bodies (LBs), the pathological hallmark of sporadic Parkinson disease (PD). Angiotensin II type 1 receptor (AT R) blocker and Rho-associated protein kinase (Rho) inhibitor have shown protective effects against 1 neurotoxins. This study examined the effects of telmisartan (AT R blocker), CGP42112 (AT R agonist) and Y1 2 27632 (Rho inhibitor) in a genetic in vitro PD model produced by α-synuclein in the H4 human neuroglioma cell line. H4 neuroglioma cells transfected with equimolar ratio of constructs encoding non-amyloid β components (NAC) (Aa 61-95) of α-synuclein carboxy-terminus fused to enhanced green fluorescent fusion protein (EGFP) (SNCA), and synphilin-1 fused to mCherry (SNCAIP) or empty vector control. Two hours later, telmisartan, CGP42112 or Y-27632 (1, 5 or 10µM) or telmisartan (5µM) + Y-27632 (5µM) was added and assayed for αsynuclein toxicity. WTSyn-EGFP-Synphilin-1 (SNCA-SNCAIP) induced marked cell toxicity compared with empty vector control. However, telmisartan (1, 5 or 10 µM) produced concentration dependent decrease in toxicity (9.07±3.51, 41.85±11.27, and 66.92±8.96%, respectively). Similarly, Y-27632 (1, 5 or 10µM) significantly reduced adenylate kinase release in a dose dependent manner (3.27±1.03, 28.54±5.64 and 46.89%, respectively) but not CGP42112. Interestingly, co-administration of telmisartan (5µM) with Y-27632 (5µM) induced synergism, producing 86.72±14.58% reduction in cytolysis. Findings from this study showed that telmisartan and Y-27632 attenuate α-synuclein pathology. This underscores the translational potential of telmisartan and Y-27632 as disease-modifying drugs for the treatment of sporadic PD. Keywords: Y-27632; telmisartan; MTT assay; adenylate kinase; cell viability; synphilin-1Abstrait La protéine α-synucléine nerveuse terminale et sa protéine synphiline-1 en interaction sont des composants protéiques majeurs des corps de Lewy (LB), caractéristique pathologique de la maladie de Parkinson (PD) sporadique. Un inhibiteur des récepteurs de l'angiotensine II de type 1 (AT1R) et un inhibiteur de la protéine kinase (Rho) associée à Rho ont montré des effets protecteurs contre les neurotoxines. Cette étude a examiné les effets du telmisartan (bloqueur AT1R), du CGP42112 (agoniste de l'AT2R) et du Y-27632 (inhibiteur de Rho) dans un modèle génétique de PD in vitro produit par la α-synucléine dans la lignée cellulaire H4 de neurogliome humain. Cellules de neurogliome H4 transfectées avec un rapport équimolaire de constructions codant pour des composants β non amyloïdes (NAC) (Aa 61-95) de l'α-synucléine carboxy-terminal fusionnée à la protéine de fusion à fluorescence verte améliorée (SNCA) et à la synphiline-1 fusionnée mCherry (SNCAIP) ou un contrôle vectoriel vide. Deux heures plus tard, le telmisartan, CGP42112 ou Y-27632 (1, 5 ou 10 µM) ou le telmisartan (5 µM) + Y-27632 (5 µM) a été ajouté et dosé pour déterminer sa toxicité en α-synucléine. WTSyn-EGFP-Synphilin-1 (SNCA-SNCAIP) a induit une toxicité cellulaire marquée par rapport au contrôle du vecteur vide. Cependant, le telmisartan (1, 5 ou 10 µM) a entraîné une diminution de la toxicité en fonction de la concentration (9,07 ± 3,51, 41,85 ± 11,27 et 66,92 ± 8,96%, respectivement). De même, le Y-27632 (1, 5 ou 10 µM) a significativement réduit la libération d'adénylate kinase de manière dépendante de la dose (3,27 ± 1,03, 28,54 ± 5,64 et 46,89%, respectivement), mais pas de CGP42112. Fait intéressant, la co-administration de telmisartan (5 µM) avec Y27632 (5 µM) a induit une synergie, produisant une réduction de 86,72 ± 14,58% de la cytolyse. Les résultats de cette étude ont montré que le telmisartan et le Y-27632 atténuent la pathologie de l'α-synucléine. Cela souligne le potentiel de traduction du telmisartan et du Y-27632 en tant que médicaments modificateurs de la maladie pour le traitement de la MP sporadique. Mots-clés: Y-27632; le telmisartan; Dosage du MTT; l'adénylate kinase; viabilité cellulaire; synphilin-1West Afr. J. Pharmacol. Drug Res. Vol.32 January – December 2017/2018; 1-

Schizophrenia risk candidate protein ZNF804A interacts with STAT2 and influences interferon-mediated gene transcription in mammalian cells

Previously evidence was presented that the single-nucleotide polymorphism rs1344706 located in an intronic region of the ZNF804A gene is associated with reduced transcript levels in fetal brains. This genetic variation in the gene encoding the zinc-finger protein ZNF804A is associated with schizophrenia (SZ) and bipolar disorder. Currently, the molecular and cellular function of ZNF804A is unclear. Here, we generated a high-confidence protein-protein interaction (PPI) network for ZNF804A using a combination of yeast two-hybrid and bioluminescence-based PPI detection assays, directly linking 12 proteins to the disease-associated target protein. Among the top hits was the signal transducer and activator of transcription 2 (STAT2), an interferon-regulated transcription factor. Detailed mechanistic studies revealed that STAT2 binds to the unstructured N-terminus of ZNF804A. This interaction is mediated by multiple short amino acid motifs in ZNF804A but not by the conserved C2H2 zinc-finger domain, which is also located at the N-terminus. Interestingly, investigations in HEK293 cells demonstrated that ZNF804A and STAT2 both co-translocate from the cytoplasm into the nucleus upon interferon (IFN) treatment. Furthermore, a concentration-dependent effect of ZNF804A overproduction on STAT2-mediated gene expression was observed using a luciferase reporter, which is under the control of an IFN-stimulated response element (ISRE). Together these results indicate the formation of ZNF804A:STAT2 protein complex and its translocation from the cytoplasm into the nucleus upon IFN stimulation, suggesting that it may function as a signal transducer that activates IFN-mediated gene expression programs

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends

Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins