Predicting direct protein interactions from affinity purification mass spectrometry data

<p>Abstract</p> <p>Background</p> <p>Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) <it>in vivo</it>. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest.</p> <p>Results</p> <p>We first propose a simple probabilistic model for the interactions captured by AP-MS experiments, under which the problem of separating direct interactions from indirect ones is formulated. Then, given idealized quantitative AP-MS data, we study the problem of identifying the most likely set of direct interactions that produced the observed data. We address this challenging graph theoretical problem by first characterizing signatures that can identify weakly connected nodes as well as dense regions of the network. The rest of the direct PPI network is then inferred using a genetic algorithm.</p> <p>Our algorithm shows good performance on both simulated and biological networks with very high sensitivity and specificity. Then the algorithm is used to predict direct interactions from a set of AP-MS PPI data from yeast, and its performance is measured against a high-quality interaction dataset.</p> <p>Conclusions</p> <p>As the sensitivity of AP-MS pipeline improves, the fraction of indirect interactions detected will also increase, thereby making the ability to distinguish them even more desirable. Despite the simplicity of our model for indirect interactions, our method provides a good performance on the test networks.</p

Identification and characterization of the BRI2 interactome in the brain

BRI family proteins are ubiquitous type II transmembrane proteins but BRI2 is highly expressed in some neuronal tissues. Possible BRI2 functions include neuronal maturation and differentiation. Protein complexes appear to be important in mediating its functions. Previously described BRI2 interactors include the Alzheimer's amyloid precursor protein and protein phosphatase 1, but clearly the identification of novel interactors provides an important tool to understand the role and function of BRI2. To this end three rat brain regions (cerebellum, hippocampus, and cerebral cortex) were processed by BRI2 immunoprecipitation;co-precipitating proteins were identified by Nano-HPLC-MS/MS. The pool of the brain regions resulted in 511 BRI2 interacting proteins (BRI2 brain interactome) of which 120 were brain specific and 49 involved in neuronal differentiation. Brain region-specific analyses were also carried out for cerebellum, hippocampus, and cerebral cortex. Several novel BRI2 interactors were identified among them DLG4/PSD-95, which is singularly important as it places BRI2 in the postsynaptic compartment. This interaction was validated as well as the interaction with GAP-43 and synaptophysin. In essence, the resulting BRI2 brain interactome, associates this protein with neurite outgrowth and neuronal differentiation, as well as synaptic signalling and plasticity. It follows that further studies should address BRI2 particularly given its relevance to neuropathological conditions

Investigating pathogen-host interactions and adaptation with network biology approaches

Serovars of the genus Salmonella are widespread enteric pathogens, causing acute inflammatory gut infections. However, a subgroup of Salmonella adapted to a systemic lifestyle instead of a mucosal one. A systems-level understanding of how molecular level changes accompanying this adaptive process potentially modify the behaviour of these invasive strains is crucial for future intervention processes, and possible treatments. In this thesis, I generated and analysed multi-layered interaction networks for 20 strains in the genus Salmonella. I collated protein-protein, transcriptional regulatory, and metabolic interaction data from low and high-throughput experiments and performed predictive measures to add further connections to the systems. The resulting networks culminated in the update to SalmoNet, the first integrated network database for Salmonella serovars. Through comparative network approaches, users can highlight elements under selection in these invasive serovars, increasing our understanding of the host adaptation process leading to their systemic lifestyle. During the last year of my PhD, I redeployed for 6 months to work on COVID-19 related research. This effort led to a systematic literature curation highlighting different cytokine responses in patients caused by SARS-CoV-2 compared to other similar viruses. I also led the effort to establish a new network resource, CytokineLink, aimed at highlighting avenues of cell-to-cell communication mediated by cytokines, to better understand inflammatory and infectious diseases. Overall, the work presented in this thesis has increased our understanding of the Salmonella host adaptation process, by highlighting specific elements under selection, while also exhibiting how network information can be created, and used for understanding such evolutionary processes

Caracterização funcional de novos complexos da BRI2 e da BRI3

Doutoramento em BiomedicinaA BRI2 e a BRI3 são proteínas transmembranares de tipo II cujas funções fisiológicas ainda não se encontram bem caracterizadas. Duas mutações autossómicas dominantes no gene BRI2 estão na origem de duas formas de demência rara: Demência Familiar Britânica e Demência Familiar Dinamarquesa. Estas partilham características clinicas e neuropatológicas com a doença de Alzheimer, nomeadamente deposição de proteínas anormais no cérebro. Apesar de nunca ter sido associada a qualquer doença, a BRI3 está enriquecida no sistema nervoso central o que sugere que possa ter uma função neuronal. Interações proteína-proteína têm adquirido uma importância crescente no estudo de funções de proteínas e vias de sinalização subjacentes. Neste trabalho, foram identificadas novas interações entre a BRI2 e a proteína fosfatase 1 (PP1), bem como entre a BRI3 com a PP1. A PP1 é a uma importante fosfatase serina/treonina presente em organismos eucariotas, onde se estima que seja capaz de catalisar a maioria dos eventos de defosforilação. Adicionalmente, determinámos a BRI2 e BRI3 como substratos da PP1, e identificámos os motivos RVxF presentes nos seus N-terminais como responsáveis pela ligação à PP1. A relevância fisiológica do complexo BRI2:PP1 foi investigada, e demonstrámos pela primeira vez que a fosforilação da BRI2, nomeadamente a defosforilação pela PP1, é um mecanismo de regulação do seu processamento proteolítico. Estabelecemos ainda uma relação entre os níveis de fosforilação da BRI2 com a diferenciação de processos neuronais, onde a fosforilação da proteína total parece estar envolvida no surgimento de processos neuronais, enquanto o fragmento Nterminal resultante do seu processamento parece estar relacionado com o elongamento e estabilização dos processos neuronais. No decorrer do trabalho, identificámos e caracterizámos os interactomas das proteínas BRI2 e BRI3 em cérebro, recorrendo a co-imunoprecipitações e identificação das proteínas por espectrometria de massa. Análise in silico do interactome da BRI2 reforçou os nossos resultados sugerindo um papel para esta proteína na diferenciação neuronal, e vários mecanismos subjacentes foram evidenciados. Adicionalmente, a análise dos interactomas localiza ambas proteínas na sinapse (pré e pós-sinapse). Esta evidência juntamente com a descoberta de interações com proteínas estruturais destes compartimentos celulares, nomeadamente a PSD-95, sugere um papel importante para ambas as proteínas em sinalização e transmissão sináptica. Assim, os nossos resultados compreendem informação importante para estudos futuros acerca da biologia das proteínas BRI2 e BRI3, e mecanismos celulares envolvidos, bem como na sua associação com o desenvolvimento de neuropatologias.BRI2 and BRI3 are type II transmembrane protein family members whose physiological functions are poorly understood. Two different autosomal dominant mutations in the BRI2 gene are responsible for the development of two rare early-onset forms of dementia: Familial British and Familial Danish dementias that share clinical and neuropathological characteristics with Alzheimer´s disease, involving abnormal proteins’ deposition in the brain. For BRI3 no disease association has been established so far, nevertheless, its enriched expression in the central nervous system strongly suggests a role for BRI3 in neuronal function. Protein-protein interactions are becoming increasingly important in the study of protein function and underlying signaling pathways. In this work, both BRI2 and BRI3 were identified as novel protein phosphatase 1 (PP1) interacting proteins. PP1 is the major Ser/Thr protein phosphatase present in eukaryotic organisms, which catalyses the majority of protein dephosphorylation events. Moreover, we were able to determine BRI2 and BRI3 as PP1 substrates, which bind to the latter through an RVxF-like motif located in their N-terminal part. The physiological relevance of BRI2:PP1 complex was pursued, and we provide for the first time evidence suggesting that BRI2 phosphorylation, namely via PP1 dephosphorylation, is a regulatory mechanism for its proteolytic processing. Further, a correlation between BRI2 phosphorylation and neurite outgrowth was established. The phosphorylation of full-length protein seems to promote the emergence of neurites whereas the increased BRI2 N-terminal processing fragment plays a role in neurites’ elongation and stabilization. Herein, we also identified and characterized the interactome of both BRI2 and BRI3 in the brain using co-immunoprecipitation followed by mass spectrometry analysis. In silico analysis of the BRI2 interactome strengthened our results suggesting a role in neuronal differentiation and some underlying mechanisms were pointed out. Moreover, interactomes’ analysis suggested the presence of both BRI2 and BRI3 at synapses (pre- and post-synapse). This finding together with their ability to form complexes with structural constituents of these compartments, namely PSD-95 suggested an important role for both proteins in synaptic signaling and synaptic transmission. Thus, our results provide valuable information for further studies on BRI2 and BRI3 biology, and potential underlying molecular mechanisms, as well as associations with neuropathologies