A Genetic-Proteomic Approach to Identify Cellular Components that Interact with HIV-1

Given the limited genetic coding capacity of HIV-1, it is reasonable to expect that the virus must interact with an extensive set of cellular factors and their complexes to complete its passage through the cell. Indeed, it is remarkable that the viral genome, comprising only about 0.0003% of the entire genetic capacity of the cell, commandeers the cellular environment to its own advantage. However, to date, only a small group of cellular proteins have been shown to be required for viral propagation. In an effort to recover and identify those host proteins that interact in complex with the viral machinery, we have developed a systematic genetic method to select derivatives that can encode a small, but potent, foreign epitope tag yet remain fully replication-competent in culture. In conjunction with a novel cryogenic methodology to capture and preserve viralhost interactions usually lost when more conventional isolation techniques are employed, we have recovered host complexes that interact specifically with each of three independently tagged HIV-1 proteins during progressive infection. Thus, the quantitative purification of the tagged viral proteins has allowed the identification of both described factors already known to interact with each of the targeted viral proteins and as well, unanticipated sets of new host proteins in complex with the virus and previously obscured from investigation. Identification and characterization of protein-protein interactions between the host and the virus will provide insight into the cellular processes expropriated by the virus to complete its life cycle

MALT1 protease function – from ubiquitin binding to substrate cleavage

The protease MALT1 has a key function in the activation of lymphocytes and the regulation of the immune response, by promoting the activation of pro-inflammatory transcription factors such as NF-B. Dysregulation of MALT1 is implicated in immunodeficiency, autoimmune diseases, lymphomagenesis and non-lymphoid malignancies. Therefore, MALT1 proteolytic activity has appeared as a possible pharmaceutical target, however, the precise mechanism of MALT1 protease activation and the role of individual substrate cleavage events remain incompletely defined. Thus, the aims of this study are to firstly elucidate the molecular mechanism of MALT1 activation and second, to explore the role of the MALT1-dependent cleavage of one particular substrate namely, A20. The protease activity of MALT1 is tightly controlled by conjugation of monoubiquitin to its third auto-inhibitory Ig-like domain, but the mechanism governing the release of the protease domain by a single ubiquitin moiety remains unknown. Here, we identify the Ig3 domain of MALT1 as a novel ubiquitin-binding domain, responsible for MALT1 monoubiquitination, which is essential for MALT1 proteolytic activity and lymphocyte activation. Furthermore, we reveal an allosteric communication from the monoubiquitination site through the protease-Ig3 interaction surface to the catalytic active site of the protease domain. One of the first substrates of MALT1 that has been identified is A20, a potent anti-inflammatory protein. A20 is a well-described negative regulator of the NF-B signaling pathway downstream of different pro-inflammatory stimuli and a regulator of cell death. Although MALT1-dependent A20 cleavage is generally thought to promote NF-B activity, the functional role of A20 cleavage remains controversial and not well defined. This is due to the fact that the originally described single cleavage site in A20 is not conserved among species and only a small proportion of cellular A20 undergoes cleavage. Here, we demonstrate that MALT1 cleaves A20 at a total of four distinct sites in B and T lymphocytes, including three novel cleavage sites with unusual sequence properties, which are conserved in the mouse and other species. The cleavage fragments lost their capacity to regulate the NF-B pathway, but are stable within the cell, suggesting that they retain an unknown physiological function in lymphocytes. Collectively, our findings provide fundamentally new insights into the mechanism of MALT1 protease activation and its cleavage site specificity, and suggest that MALT1-dependent A20 cleavage has roles that go beyond the enhancement of NF-B activity. -- La protéase MALT1 a une fonction clé dans l’activation des lymphocytes et la régulation de la réponse immunitaire, en favorisant l’activation de facteurs de transcription pro- inflammatoires comme le NF-B. La dérégulation de MALT1 est impliquée dans l’immunodéficience, les maladies auto-immunes, la lymphomagénèse et les tumeurs malignes non-lymphoides. Par conséquent, l’activité protéolytique de MALT1 est apparue comme une cible thérapeutique possible, cependant le mécanisme précis de l’activation de la protéase MALT1 et le rôle du clivage de chacun de ses substrats restent en partie incompris. Ainsi les objectifs de cette étude sont d’abord d’élucider le mécanisme moléculaire d’activation de MALT1 puis d’explorer le rôle du clivage dépendant de MALT1 d’un substrat en particulier nommé A20. L’activité protéase de MALT1 est étroitement contrôlée par la conjugaison d’une monoubiquitine à son troisième domaine auto-inhibiteur de type Ig, mais le mécanisme régissant la libération du domaine protéase par un seul fragment d'ubiquitine reste inconnu. Ici, nous avons identifié le domaine Ig3 de MALT1 comme un nouveau domaine de liaison à l’ubiquitine, responsable de la monoubiquitination de MALT1, qui est essentielle pour son activité protéolytique et l’activation lymphocytaire. De plus, nous avons révélé une transmission allostérique du site de monoubiquitination à travers la surface d’interaction protéase-Ig3 au site catalytique actif du domaine protéase. L’un des premiers substrats de MALT1 qui a été identifié est A20, une protéine anti-inflammatoire puissante. A20 est un régulateur négatif bien décrit de la voie de signalisation NF-B en aval de différents stimuli pro-inflammatoires et un régulateur de la mort cellulaire. Bien que le clivage de A20 dépendant de MALT1 soit généralement vu comme un promoteur de l’activité de NF-B, le rôle fonctionnel du clivage de A20 reste controversé et mal défini. Cela est dû au fait que le site de clivage originellement découvert de A20 n’est pas conservé chez les autres espèces et que seulement une petite partie de la protéine cellulaire est clivée. Ici, nous démontrons que MALT1 clive A20 sur un total de quatre sites distincts dans les lymphocytes B et T. Nous avons également identifié trois nouveaux sites de clivage avec des motifs inhabituels, sites qui sont conservés chez la souris et d’autres espèces. Les fragments issus du clivage ont perdu leur capacité à réguler la voie NF-B, mais sont stables dans la cellule, suggérant qu’ils gardent une fonction physiologique inconnue dans les lymphocytes. Ensemble, nos résultats apportent de nouvelles informations fondamentales sur le mécanisme d’activation de la protéase MALT1 et sur la spécificité de ses sites de clivage, et suggèrent que le clivage de A20 dépendant de MALT1 a un rôle qui va au- delà d’une simple activation de NF-B

Examining the mechanistic regulation of starvation-induced autophagy via the identification and characterisation of novel ULK kinase substrates

Autophagy involves the formation of an endoplasmic reticulum-derived membrane termed a phagophore which expands to engulf cytoplasmic cargo before sealing to form an autophagosome. Amino acid starvation is amongst the most potent autophagic stimuli, however whilst the key signalling complexes involved in starvation-induced autophagy are known, the precise regulatory mechanisms remain poorly understood. The serine/threonine kinase ULK1 and close homolog ULK2 assume the most upstream position in the autophagic signalling cascade and play a crucial yet enigmatic role in coordinating the autophagic machinery. To further understand the mechanisms of starvation-induced autophagy, I performed a number of unbiased phosphoproteomic screens to identify ULK substrates before classifying their roles in starvation-induced autophagy. Analysis of these datasets has revealed that loss of ULK results in significant changes to the phosphoproteome and has yielded a high confidence list of potential substrates whilst also offering interesting insights into the veracity of the published ULK consensus signature. Amongst the novel phosphorylation targets are components of the retromer and AMPK complexes along with multiple components of the class III PI3K VPS34 complex. The pseudokinase p150, scaffolding component of the VPS34 complex, is phosphorylated by ULK1 in vitro and in vivo at serine 861. CRISPR-based knockout of p150 results in inhibition of autophagy and endosomal trafficking, whilst mutating the phosphorylated residue in p150 alters both omegasome establishment and autophagic flux. Furthermore, incorporation of phosphomutant p150 into the VPS34 complex modulates its lipid kinase activity in vitro. These data identify a novel ULK-dependent signalling axis and help illuminate the complexities of signal transduction in autophagy