Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

eIF4A inactivates TORC1 in response to amino acid starvation.

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. We identify here the eIF4A-containing eIF4F translation initiation complex as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila We find that TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal.

Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.

Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization.

The use of time series profiling to identify groups of functionally related genes (synexpression groups) is a powerful approach for the discovery of gene function. Here we apply this strategy during Ras(V12) immortalization of Drosophila embryonic cells, a phenomenon not well characterized. Using high-resolution transcriptional time-series datasets, we generated a gene network based on temporal expression profile similarities. This analysis revealed that common immortalized cells are related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells. Remarkably, the immortalized cells retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. Further, we validated in vivo the transcription factor CG9650, the ortholog of mammalian Bcl11a/b, as a regulator of AMP proliferation predicted by our analysis. Our study demonstrates the power of time series synexpression analysis to characterize Drosophila embryonic progenitor lines and identify stem/progenitor cell regulators.

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.

Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells.

Immune signalling pathways need to be tightly regulated as overactivation of these pathways can result in chronic inflammatory diseases and cancer. NF-κB signalling and associated innate immune pathways are crucial in the first line of defense against infection in all animals. In a genome-wide RNAi screen for modulators of Drosophila immune deficiency (IMD)/NF-κB signalling, we identified components of the Ras/MAPK pathway as essential for suppression of IMD pathway activity, even in the absence of an immune challenge. Downregulation of Ras/MAPK activity mimics the induction of innate immune responses by microbial patterns. Conversely, ectopic Ras/MAPK pathway activation results in the suppression of Drosophila IMD/NF-κB signalling. Mechanistically, we show that the Ras/MAPK pathway acts by inducing transcription of the IMD pathway inhibitor Pirk/Rudra/PIMS. Finally, in vivo experiments demonstrate a requirement for Ras/MAPK signalling in restricting innate immune responses in haemocytes, fat body and adult intestinal stem cells. Our observations provide an example of a pathway that promotes cell proliferation and has simultaneously been utilized to limit the immune response.

p24 proteins are required for secretion of Wnt ligands.

During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.

N-terminal proteoforms may engage in different protein complexes.

Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.