Frequent Assembly of Chimeric Complexes in the Protein Interaction Network of an Interspecies Yeast Hybrid.

Hybrids between species often show extreme phenotypes, including some that take place at the molecular level. In this study, we investigated the phenotypes of an interspecies diploid hybrid in terms of protein-protein interactions inferred from protein correlation profiling. We used two yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum, which are interfertile, but yet have proteins diverged enough to be differentiated using mass spectrometry. Most of the protein-protein interactions are similar between hybrid and parents, and are consistent with the assembly of chimeric complexes, which we validated using an orthogonal approach for the prefoldin complex. We also identified instances of altered protein-protein interactions in the hybrid, for instance, in complexes related to proteostasis and in mitochondrial protein complexes. Overall, this study uncovers the likely frequent occurrence of chimeric protein complexes with few exceptions, which may result from incompatibilities or imbalances between the parental proteomes.

Yeast proteins do not practice social distancing as species hybridize.

With the current COVID-19 pandemic, we all realized how important interactions are. Interactions are everywhere. At the cellular level, protein interactions play a key role and their ensemble, also called interactome, is often referred as the basic building blocks of life. Given its importance, the maintenance of the integrity of the interactome is a real challenge in the cell. Many events during evolution can disrupt interactomes and potentially result in different characteristics for the organisms. However, the molecular underpinnings of changes in interactions at the cellular level are still largely unexplored. Among the perturbations, hybridization puts in contact two different interactomes, which may lead to many changes in the protein interaction network of the hybrid, including gains and losses of interactions. We recently investigated the fate of the interactomes after hybridization between yeast species using a comparative proteomics approach. A large-scale conservation of the interactions was observed in hybrids, but we also noticed the presence of proteostasis-related changes. This suggests that, despite a general robustness, small differences may accumulate in hybrids and perturb their protein physiology. Here, we summarize our work with a broader perspective on the importance of interactions.

Synaptojanin-2 binding protein stabilizes the Notch ligands DLL1 and DLL4 and inhibits sprouting angiogenesis.

RATIONALE: The formation of novel blood vessels is initiated by vascular endothelial growth factor. Subsequently, DLL4-Notch signaling controls the selection of tip cells, which guide new sprouts, and trailing stalk cells. Notch signaling in stalk cells is induced by DLL4 on the tip cells. Moreover, DLL4 and DLL1 are expressed in the stalk cell plexus to maintain Notch signaling. Notch loss-of-function causes formation of a hyperdense vascular network with disturbed blood flow. OBJECTIVE: This study was aimed at identifying novel modifiers of Notch signaling that interact with the intracellular domains of DLL1 and DLL4. METHODS AND RESULTS: Synaptojanin-2 binding protein (SYNJ2BP, also known as ARIP2) interacted with the PDZ binding motif of DLL1 and DLL4, but not with the Notch ligand Jagged-1. SYNJ2BP was preferentially expressed in stalk cells, enhanced DLL1 and DLL4 protein stability, and promoted Notch signaling in endothelial cells. SYNJ2BP induced expression of the Notch target genes HEY1, lunatic fringe (LFNG), and ephrin-B2, reduced phosphorylation of ERK1/2, and decreased expression of the angiogenic factor vascular endothelial growth factor (VEGF)-C. It inhibited the expression of genes enriched in tip cells, such as angiopoietin-2, ESM1, and Apelin, and impaired tip cell formation. SYNJ2BP inhibited endothelial cell migration, proliferation, and VEGF-induced angiogenesis. This could be rescued by blockade of Notch signaling or application of angiopoietin-2. SYNJ2BP-silenced human endothelial cells formed a functional vascular network in immunocompromised mice with significantly increased vascular density. CONCLUSIONS: These data identify SYNJ2BP as a novel inhibitor of tip cell formation, executing its functions predominately by promoting Delta-Notch signaling.

Mobile App Prototype in Older Adults for Postfracture Acute Pain Management: User-Centered Design Approach.

BACKGROUND: Postfracture acute pain is often inadequately managed in older adults. Mobile health (mHealth) technologies can offer opportunities for self-management of pain; however, insufficient apps exist for acute pain management after a fracture, and none are designed for an older adult population. OBJECTIVE: This study aims to design, develop, and evaluate an mHealth app prototype using a human-centered design approach to support older adults in the self-management of postfracture acute pain. METHODS: This study used a multidisciplinary and user-centered design approach. Overall, 7 stakeholders (ie, 1 clinician-researcher specialized in internal medicine, 2 user experience designers, 1 computer science researcher, 1 clinical research assistant researcher, and 2 pharmacists) from the project team, together with 355 external stakeholders, were involved throughout our user-centered development process that included surveys, requirement elicitation, participatory design workshops, mobile app design and development, mobile app content development, and usability testing. We completed this study in 3 phases. We analyzed data from prior surveys administered to 305 members of the Canadian Osteoporosis Patient Network and 34 health care professionals to identify requirements for designing a low-fidelity prototype. Next, we facilitated 4 participatory design workshops with 6 participants for feedback on content, presentation, and interaction with our proposed low-fidelity prototype. After analyzing the collected data using thematic analysis, we designed a medium-fidelity prototype. Finally, to evaluate our medium-fidelity prototype, we conducted usability tests with 10 participants. The results informed the design of our high-fidelity prototype. Throughout all the phases of this development study, we incorporated inputs from health professionals to ensure the accuracy and validity of the medical content in our prototypes. RESULTS: We identified 3 categories of functionalities necessary to include in the design of our initial low-fidelity prototype: the need for support resources, diary entries, and access to educational materials. We then conducted a thematic analysis of the data collected in the design workshops, which revealed 4 themes: feedback on the user interface design and usability, requests for additional functionalities, feedback on medical guides and educational materials, and suggestions for additional medical content. On the basis of these results, we designed a medium-fidelity prototype. All the participants in the usability evaluation tests found the medium-fidelity prototype useful and easy to use. On the basis of the feedback and difficulties experienced by participants, we adjusted our design in preparation for the high-fidelity prototype. CONCLUSIONS: We designed, developed, and evaluated an mHealth app to support older adults in the self-management of pain after a fracture. The participants found our proposed prototype useful for managing acute pain and easy to interact with and navigate. Assessment of the clinical outcomes and long-term effects of our proposed mHealth app will be evaluated in the future.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of IκBα and nuclear accumulation of NF-κB.

Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulate the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-κB. Furthermore phosphorylation and degradation of the inhibitor protein IκBα and nuclear accumulation of p65 and p50 NF-κB proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored IκBα and NF-κB activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.

Gene duplication can impart fragility, not robustness, in the yeast protein interaction network.

The maintenance of duplicated genes is thought to protect cells from genetic perturbations, but the molecular basis of this robustness is largely unknown. By measuring the interaction of yeast proteins with their partners in wild-type cells and in cells lacking a paralog, we found that 22 out of 56 paralog pairs compensate for the lost interactions. An equivalent number of pairs exhibit the opposite behavior and require each other's presence for maintaining their interactions. These dependent paralogs generally interact physically, regulate each other's abundance, and derive from ancestral self-interacting proteins. This reveals that gene duplication may actually increase mutational fragility instead of robustness in a large number of cases.

Expression Profiling of Ribosome Biogenesis Factors Reveals Nucleolin as a Novel Potential Marker to Predict Outcome in AML Patients.

Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

Multi-scale perturbations of protein interactomes reveal their mechanisms of regulation, robustness and insights into genotype-phenotype maps.

Cellular architectures and signaling machineries are organized through protein-protein interactions (PPIs). High-throughput methods to study PPIs in yeast have opened a new perspective on the organization of the cell by allowing the study of whole protein interactomes. Recent investigations have moved from the description of this organization to the analysis of its dynamics by experimenting how protein interaction networks (PINs) are rewired in response to perturbations. Here we review studies that have used the budding yeast as an experimental system to explore these altered networks. Given the large space of possible PPIs and the diversity of potential genetic and environmental perturbations, high-throughput methods are an essential requirement to survey PIN perturbations on a large scale. Network perturbations are typically conceptualized as the removal of entire proteins (nodes), the modification of single PPIs (edges) or changes in growth conditions. These studies have revealed mechanisms of PPI regulation, PIN architectural organization, robustness and sensitivity to perturbations. Despite these major advances, there are still inherent limits to current technologies that lead to a trade-off between the number of perturbations and the number of PPIs that can be considered simultaneously. Nevertheless, as we exemplify here, targeted approaches combined with the existing resources remain extremely powerful to explore the inner organization of cells and their responses to perturbations.

The roles of nucleolin subcellular localization in cancer.

Nucleolin (NCL) is one of the most abundant non ribosomal protein of the nucleolus where it plays a central role in polymerase I transcription. NCL is also found outside of the nucleolus, in the nucleoplasm, cytoplasm as well as on the cell membrane. It acts in all cell compartments to control cellular homeostasis and therefore each cellular pool of NCL can play a different role in cancer development. NCL overexpression and its increased localization at the cell membrane is a common feature of several tumor cells. In cancer cells, NCL overexpression influences cell survival, proliferation and invasion through its action on different cellular pathways. In this review, we describe how the multiple functions of NCL that are associated to its multiple cellular localization can participate to the development of cancer.

Soluble Notch ligand and receptor peptides act antagonistically during angiogenesis.

AIMS: Notch signalling is essential for blood vessel formation. During angiogenesis, the Notch ligand DLL4 on the leading tip cell activates Notch receptors on the adjacent stalk cells. DLL4-Notch signalling is impaired by the Notch ligand JAG1 in endothelial cells. The Delta/Serrate/Lag2 (DSL) domain of the Notch ligands binds to the EGF-like repeats 11-13 of the Notch receptor. This study aimed to elucidate how soluble proteins containing these short domains interfere with Notch signalling during angiogenesis. METHODS AND RESULTS: Adenoviral vectors were generated to express the DSL domains of DLL1, DLL4, JAG1, and the Notch1 EGF-like repeats 11-13 fused to immunoglobulin-G heavy chain. These soluble ligand peptides inhibited Notch signalling in endothelial cells and this caused hyperbranching in cellular angiogenesis assays and in the neonatal mouse retina. The soluble Notch receptor peptides bound stronger to JAG1 than DLL4 ligands, resulting in increased signalling activity. This led to impaired tip cell formation and less vessel sprouting in the retina. CONCLUSION: The minimal binding domains of Notch ligands are sufficient to interfere with Notch signalling. The corresponding soluble Notch1 EGF11-13 peptide binds stronger to inhibitory Notch ligands and thereby promotes Notch signalling in endothelial cells.