The identification of small proteins and proteins produced from unannotated open reading frames (called alternative proteins or AltProts) has changed our vision of the proteome and has attracted more and more attention from the scientific community. Despite several studies investigating particular AltProts in diseases and demonstrating their importance in such context, we are still missing data on their expression and functions in many pathologies. Among these, pancreatic ductal adenocarcinoma (PDAC) is a particularly relevant case to study alternative proteins. Indeed, late detection of this disease, notably due to the lack of reliable biomarkers of early-stage PDAC, and the fact that tumors rapidly develop resistance to most of the treatments used in the clinics warrant the exploration of new repertoires of molecules. In the present article, we aim to investigate the alternative proteome of pancreatic cancer cell lines as a first attempt to decipher the expression of AltProts in PDAC. Thanks to a combined data-dependent and data-independent acquisition mass spectrometry workflow, we were able to identify tryptic peptides matching 113 AltProts in a panel of 6 cell lines. In addition, we identified AltProts differentially expressed between pancreatic cancer cell lines and other cells (HeLa and HEK293T). Finally, mining the TCGA and Gtex databases showed that the corresponding transcripts encoding several AltProts we identified are differentially expressed between PDAC tumors and normal tissues and are correlated with the patient’s survival.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes responsible for linking a transfer RNA (tRNA) with its cognate amino acid present in all the kingdoms of life. Besides their aminoacyl-tRNA synthetase activity, it was described that many of these enzymes can carry out non-canonical functions. They were shown to be involved in important biological processes such as metabolism, immunity, development, angiogenesis and tumorigenesis. In the present work, we provide evidence that tryptophanyl-tRNA synthetase might be involved in a negative feedback loop mitigating the expression of certain interferon-γ-induced genes. Mining the available TCGA and Gtex data, we found that WARS was highly expressed in cutaneous melanoma (SKCM) compared to other cancers and is of good prognosis for this particular cancer type. WARS expression correlates with genes involved in antigen processing and presentation but also transcription factors involved in IFN-γ signaling such as STAT1. In addition, WARS was found in complex with STAT1 in A375 cells treated with IFN-γ. Finally, we showed that knocking down WARS expression during IFN-γ stimulation further increases the expression of GBP2, APOL1, ISG15, HLA-A and IDO1.

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found – using transcriptomic and proteomic analyses – that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3—a mediator of YWF sensing upstream of mTOR—is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular “satiety center” carries therefore potential therapeutic implications for cancer.

Supplementary Table S2: List of SPANX-interacting proteins identified by LC-MS/MS

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

Supplementary Movie S1 - 3D representation around the Y axis of A375 cell nucleus

Supplementary Table S1: Clinical information related to the PDX used in Fig1A

The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasm-a response regulated by newly identified mTOR-agonistic amino acids-Tyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.

Supplementary Table S3: List of the genes that were identified by RNAseq to be significantly deregulated upon SPANX KD.

Supplemental Methods, Figures, Figure legends, Table and Movie Legends

Supplementary Movie S2 - 3D representation around the X axis of A375 cell nucleus

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9–driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.

Short open reading frame (sORF)–encoded polypeptides (SEPs) have recently emerged as key regulators of major cellular processes. Computational methods for the annotation of sORFs combined with transcriptomics and ribosome profiling approaches predicted the existence of tens of thousands of SEPs across the kingdom of life. Although, we still lack unambiguous evidence for most of them. The method of choice to validate the expression of SEPs is mass spectrometry (MS)-based peptidomics. Peptides are less abundant than proteins, which tends to hinder their detection. Therefore, optimization and enrichment methods are necessary to validate the existence of SEPs. In this article, we discuss the challenges for the detection of SEPs by MS and recent developments of biochemical approaches applied to the study of these peptides. We detail the advances made in the different key steps of a typical peptidomics workflow and highlight possible alternatives that have not been explored yet.

ABSTRACTAcute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we found that increased expression and abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patients correlated with poor prognosis. RNF5 inhibition decreased AML cell growth in culture andin vivo, and blocked development of MLL-AF9–driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition led to transcriptional changes that overlapped with those seen upon HDAC1 inhibition. RNF5 induced the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment and subsequent epigenetic regulation of genes involved in AML development and maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhanced the sensitivity of AML cells to histone deacetylase (HDAC) inhibitors. Notably, low expression ofRNF5andHDACcoincided with a favorable prognosis. Our studies identified ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML while highlighting RNF5/RBBP4 as markers to stratify patients for treatment with HDAC inhibitors.

The NF-κB transcription factor is involved in inflammation and cell proliferation, survival, and transformation. It is a heterodimer made of p50 or p52 and a member of the Rel family of proteins. p50 and p52 are derived from limited ubiquitin- and proteasome-mediated proteolytic processing of the larger precursors p105 and p100, respectively. Both precursors can be either processed or completely degraded by the ubiquitin-proteasome system. Previous work in our laboratory identified KPC1 as a ubiquitin ligase that mediates processing of p105 to the p50 subunit. Overexpression of the ligase leads to increased level of p50 with a resultant marked tumor-suppressive effect. In the present study, we identify FBXO7, a known ubiquitin ligase that binds to p105 and ubiquitinates it, but surprisingly, leads to its accumulation and to that of p65 - the Rel partner of p50 - and to increased cell proliferation. Importantly, a ΔF-Box mutant of FBXO7 which is inactive has similar effects on accumulation of p105 and cell proliferation, strongly suggesting that p105 is a pseudo substrate of FBXO7.

AbstractTheDrosophila shaggy(sgg) gene encodes the major fly orthologue of Glycogen Synthase Kinase −3 (GSK-3), a key highly conserved kinase at the heart of many signalling pathways. Thesgglocus is complex, encoding multiple protein isoforms that are expressed in distinct temporal and tissue-specific patterns across development. Its isoforms predominantly differ at the carboxy and amino termini due to the use of different transcriptional start sites and alternative splicing events that include internal and terminal exons. One interesting class of proteins isoforms is represented by the Sgg-PD class (Sgg46), three proteoforms that contain a large 582 amino acid N-terminal domain which contains recognition sites for caspase-mediated cleavage. Regulated cleavage at these sites by non-apoptotic caspases has previously been implicated in the regulation of Sgg activity in adult bristle development. Here, we take a genome engineering approach to introduce specific tags into this unique Sgg-PD exon and utilise these for localisation and protein interaction studies. We also generated new loss of function alleles and specific mutations in the caspase cleavage motifs. We find that loss of functions Sgg-PD class alleles are viable and fertile, but exhibit adult locomotor and bristle defects. Expression analysis of lines carrying tags on both sides of the caspase cleavage sites indicates that the cleavage is developmentally regulated during embryogenesis. Surprisingly, we found that in some cells, particularly embryonic hemocytes, the N-terminal domain released by caspase cleavage is retained while the polypeptide containing the conserved kinase domain is apparently lost. Transcriptomic analysis of embryos homozygous for the new caspase-insensitive allele indicates a role for Sgg-PD in the regulation of cytoskeletal and cell junction functions, which is supported by proteomics analysis using specific in locus tags to identify common and unique protein interaction partners with N- and C-terminal domains. Taken together, our work identifies new activities for the Sgg protein and uncovers unexpected roles for caspase cleavage in Sgg biology.

Abstract Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. Implications: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

216 Cancer/testis antigens (CTA) are tumor antigens whose expression is normally restricted to the testis. More than half of CTA genes are located on the X chromosome and form a branch called X-CTA [1]. Unlike the remaining CTA genes located throughout the genome, multigene X-CTA families are present as clusters on the X chromosome. Among them, the Sperm Protein Associated with the Nucleus on the X chromosome (SPANX) family is composed by five members [SPANX-A1, -A2 (-A1 and -A2 being two copies of the same gene), -B1, -C and -D]. As SPANX proteins are highly homologous and cannot be distinguished by antibody-based techniques, unless stated otherwise, hereafter we will use the term “SPANX” to refer to these five proteins. SPANX proteins are expressed post-meiosis in round and elongating spermatids [2], and their expression positively correlates with male fertility measured by pregnancy rate [3]. SPANX function in spermatids and spermatozoa is poorly characterized. However, cell fractionation analysis revealed that SPANX is abnormally distributed in samples with low spermatozoa motility [4] suggesting that SPANX is related to this process. Furthermore, identification of SPANX partners in spermatozoa has revealed proteins functioning in nuclear organization, mitochondrial metabolism and flagellar motility [5]. The discovery that SPANX genes are expressed in tumor cells was reported in a search for metastasis-specific genes in the melanoma line 1F6m, a metastatic variant of the parental 1F6 line [6]. Since then, SPANX gene expression has been observed in numerous malignancies, including breast cancer and hematological malignancies, as well as melanoma [6,7].