Microscopic marvels: Decoding the role of micropeptides in innate immunity.

The innate immune response is under selection pressures from changing environments and pathogens. While sequence evolution can be studied by comparing rates of amino acid mutations within and between species, how a gene's birth and death contribute to the evolution of immunity is less known. Short open reading frames, once regarded as untranslated or transcriptional noise, can often produce micropeptides of <100 amino acids with a wide array of biological functions. Some micropeptide sequences are well conserved, whereas others have no evolutionary conservation, potentially representing new functional compounds that arise from species-specific adaptations. To date, few reports have described the discovery of novel micropeptides of the innate immune system. The diversity of immune-related micropeptides is a blind spot for gene and functional annotation. Immune-related micropeptides represent a potential reservoir of untapped compounds for understanding and treating disease. This review consolidates what is currently known about the evolution and function of innate immune-related micropeptides to facilitate their investigation.

Peptidomics and machine-learning-based evaluation of ncRNA-derived micropeptides in breast cancer: Expression patterns and functional/therapeutic insights.

Breast cancer is a highly heterogeneous disease characterized by different subtypes arising from molecular alterations that give the disease different phenotypes, clinical behaviors, and prognostic. The ncRNA-derived micropeptides (MPs) represent a novel layer of complexity in cancer study once they can be biologically active and can present potential as biomarkers and also in therapeutics. However, few large-scale studies address the expression of these peptides at the peptidomics level or evaluate their functions and potential in peptide-based therapeutics for breast cancer. In this study, we propose deepening the landscape of ncRNA-derived MPs in breast cancer subtypes and advance the comprehension of the relevance of these molecules to the disease. Firstly, we constructed a 16,349 unique putative MP sequence dataset by integrating two previously published lists of predicted ncRNA-derived MPs. We evaluated its expression on high-throughput mass spectrometry data of breast tumor samples from different subtypes. Next, we applied several machine and deep learning tools, such as AntiCP 2.0, MULocDeep, PEPstrMOD, Peptipedia, and PreAIP, to predict its functions, cellular localization, tertiary structure, physicochemical features, and other properties related to therapeutics. We identified 58 peptides expressed on breast tissue, including 27 differentially expressed MPs in tumor compared to non-tumor samples and MPs exhibiting tumor or subtype specificity. These peptides presented physicochemical features compatible with the canonical proteome and were predicted to influence the tumor immune environment and participate in cell communication, metabolism, and signaling processes. Also, some MPs presented potential as anti-cancer, anti-inflammatory, and anti-angiogenic molecules. Our data demonstrate that MPs derived from ncRNAs have expression patterns associated with specific breast cancer subtypes and tumor specificity, thus highlighting their potential as biomarkers for molecular classification. We also reinforce the relevance of MPs as biologically active molecules that play a role in breast tumorigenesis, besides their potential in peptide-based therapeutics.

Microproteins transitioning into a new Phase: Defining the undefined.

Recent advancements in omics technologies have unveiled a hitherto unknown group of short polypeptides called microproteins (miPs). Despite their size, accumulating evidence has demonstrated that miPs exert varied and potent biological functions. They act in paracrine, juxtracrine, and endocrine fashion, maintaining cellular physiology and driving diseases. The present study focuses on biochemical and biophysical analysis and characterization of twenty-four human miPs using distinct computational methods, including RIDAO, AlphaFold2, D2P2, FuzDrop, STRING, and Emboss Pep wheel. miPs often lack well-defined tertiary structures and may harbor intrinsically disordered regions (IDRs) that play pivotal roles in cellular functions. Our analyses define the physicochemical properties of an essential subset of miPs, elucidating their structural characteristics and demonstrating their propensity for driving or participating in liquid-liquid phase separation (LLPS) and intracellular condensate formation. Notably, miPs such as NoBody and pTUNAR revealed a high propensity for LLPS, implicating their potential involvement in forming membrane-less organelles (MLOs) during intracellular LLPS and condensate formation. The results of our study indicate that miPs have functionally profound implications in cellular compartmentalization and signaling processes essential for regulating normal cellular functions. Taken together, our methodological approach explains and highlights the biological importance of these miPs, providing a deeper understanding of the unusual structural landscape and functionality of these newly defined small proteins. Understanding their functions and biological behavior will aid in developing targeted therapies for diseases that involve miPs.

Non-Mature miRNA-Encoded Micropeptide miPEP166c Stimulates Anthocyanin and Proanthocyanidin Synthesis in Grape Berry Cells.

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.

Small open reading frames: a comparative genetics approach to validation.

Open reading frames (ORFs) with fewer than 100 codons are generally not annotated in genomes, although bona fide genes of that size are known. Newer biochemical studies have suggested that thousands of small protein-coding ORFs (smORFs) may exist in the human genome, but the true number and the biological significance of the micropeptides they encode remain uncertain. Here, we used a comparative genomics approach to identify high-confidence smORFs that are likely protein-coding. We identified 3,326 high-confidence smORFs using constraint within human populations and evolutionary conservation as additional lines of evidence. Next, we validated that, as a group, our high-confidence smORFs are conserved at the amino-acid level rather than merely residing in highly conserved non-coding regions. Finally, we found that high-confidence smORFs are enriched among disease-associated variants from GWAS. Overall, our results highlight that smORF-encoded peptides likely have important functional roles in human disease.

Micropeptides Identified from Human Genomes.

Advanced analytic techniques, such as ribosome profiling and mass spectrometry, as well as improved bioinformatics technology, have promoted the field of genome annotation forward and have identified thousands of likely coding short open reading frames (sORFs) in the human genome. The discovery of sORFs and their products allows us to realize that the complexity of the human genome is far greater than previously assumed. Here, we provide a review of human micropeptides encoded by various transcripts such as mitochondrial rRNAs, long noncoding RNAs, circular RNAs, upstream of mRNAs, and so on.

Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.

BACKGROUND: Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma. METHODS: The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways. RESULTS: The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects. CONCLUSIONS: This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.

Regulation of small RNA-mediated high temperature stress responses in crop plants.

KEY MESSAGE: Small RNAs have emerged as key players of gene expression regulation. Several lines of evidences highlight their role in modulating high temperature stress responsiveness in plants. Throughout their life cycle, plants have to regulate their gene expression at various developmental phases, physiological changes, and in response to biotic or environmental stress. High temperature is one the most common abiotic stress for crop plants, that results in impaired morphology, physiology, and yield. However, plants have certain mechanisms that enable them to withstand such conditions by modulating the expression of stress-related genes. Small RNA (sRNA)-regulated gene expression is one such mechanism which is ubiquitous in all eukaryotes. The sRNAs mainly include micro RNAs (miRNAs) and small interfering RNAs (siRNAs). They are primarily associated with the gene silencing either through translation inhibition, mRNA degradation, or DNA methylation. During high temperature stress the increased or decreased level of miRNAs altered the protein accumulation of target transcripts and, therefore, regulate stress responses. Several reports are available in plants which are genetically engineered through expressing artificial miRNAs resulted in thermotolerance. sRNAs have also been reported to bring the epigenetic changes on chromatin region through RNA-dependent DNA methylation (RdDM). The present article draws a brief illustration of sRNA origin, their functional mechanisms, role in high temperature stress, and possible application for developing stress tolerant crop plants.

Research progress on high throughput discovery and functional verification of functional micropeptides.

With the rapid development of computational biology and deep sequencing technology, more and more studies have shown that a large number of non-classical open reading frames that have not been annotated and hidden in non-coding RNA can encode functional micropeptide. This article reviewed the current research status and technology strategy of gene sources, biological properties, predicted methods and functional verification of micropeptide, providing theoretical and reference basis for the subsequent discovery of micropeptides, research on regulatory mechanisms and development of novel targets and biomarkers.

Protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the SERCA calcium transporter.

The transport activity of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) in cardiac myocytes is modulated by an inhibitory interaction with a transmembrane peptide, phospholamban (PLB). Previous biochemical studies have revealed that PLB interacts with a specific inhibitory site on SERCA, and low-resolution structural evidence suggests that PLB interacts with distinct alternative sites on SERCA. High-resolution details of the structural determinants of SERCA regulation have been elusive because of the dynamic nature of the regulatory complex. In this study, we used computational approaches to develop a structural model of SERCA-PLB interactions to gain a mechanistic understanding of PLB-mediated SERCA transport regulation. We combined steered molecular dynamics and membrane protein-protein docking experiments to achieve both a global search and all-atom force calculations to determine the relative affinities of PLB for candidate sites on SERCA. We modeled the binding of PLB to several SERCA conformations, representing different enzymatic states sampled during the calcium transport catalytic cycle. The results of the steered molecular dynamics and docking experiments indicated that the canonical PLB-binding site (comprising transmembrane helices M2, M4, and M9) is the preferred site. This preference was even more stringent for a superinhibitory PLB variant. Interestingly, PLB-binding specificity became more ambivalent for other SERCA conformers. These results provide evidence for polymorphic PLB interactions with novel sites on M3 and with the outside of the SERCA helix M9. Our findings are compatible with previous physical measurements that suggest that PLB interacts with multiple binding sites, conferring dynamic responsiveness to changing physiological conditions.

Evidence That Regulation of Pri-miRNA/miRNA Expression Is Not a General Rule of miPEPs Function in Humans.

Some miRNAs are located in RNA precursors (pri-miRNAs) annotated as long non-coding (lncRNAs) due to absence of long open reading frames (ORFs). However, recent studies have shown that some lnc pri-miRNAs encode peptides called miPEPs (miRNA-encoded peptides). Initially discovered in plants, three miPEPs have also been identified in humans. Herein, we found that a dozen human pri-miRNAs potentially encode miPEPs, as revealed by ribosome profiling and proteomic databases survey. So far, the only known function of plant miPEPs is to enhance the transcription of their own pri-miRNAs, thereby increasing the level and activity of their associated miRNAs and downregulating the expression of their target genes. To date, in humans, only miPEP133 was shown to promote a positive autoregulatory loop. We investigated whether other human miPEPs are also involved in regulating the expression of their miRNAs by studying miPEP155, encoded by the lnc MIR155HG, miPEP497, a sORF-encoded peptide within lnc MIR497HG, and miPEP200a, encoded by the pri-miRNA of miR-200a/miR-200b. We show that overexpression of these miPEPs is unable to impact the expression/activity of their own pri-miRNA/miRNAs in humans, indicating that the positive feedback regulation observed with plant miPEPs and human miPEP133 is not a general rule of human miPEP function.

[Progress on sORF-encoded micropeptides].

Existing research has shown that there are a large amount of non-coding RNAs (ncRNAs) in organisms. Short open reading frames (sORFs) abundantly exist in molecular sequences inaccurately annotated as ncRNAs. Several sORFs can be transcribed and translated into evolutionarily conserved micropeptides, which were ignored in previous studies due to short sequence lengths and the limitations of research techniques. To date, sORF-encoded micropeptides with various functions have been found to play important roles in regulating vital biological activities. This article reviews the functional micropeptides which have been found in recent years, introduces the new micropeptide designated as MIAC that we have discovered and describes the related technologies for mining potential micropeptides, thereby providing insights and references for new micropeptide discovery for researchers.

SERCA2a: a key protein in the Ca2+ cycle of the heart failure.

Calcium ion (Ca2+) cycle plays a crucial role in the contraction and relaxation of cardiomyocytes. The sarcoplasmic reticulum (SR) acts as an organelle for storing Ca2+, which mediated the release and re-uptake of Ca2+ during contraction and relaxation. Disorders of SR function lead to the dysfunction of Ca2+ cycle and myocardial cell function. The sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) acts as a subtype of SERCA expressed in the heart, which mediates the contraction of cardiomyocytes and Ca2+ in the cytoplasm to re-enter into the SR. The rate of uptake of Ca2+ by the SR determines the rate of myocardial relaxation. The regulation of SERCA2a activity controls the contractility and relaxation of the heart, affecting cardiac function. The expression and activity of SERCA2a are reduced in failing hearts. Gene therapy by increasing the expression of SERCA2a in the heart has been proven effective. In addition, SERCA2a is regulated by a variety of factors, including transmembrane micropeptides, protein kinases, and post-translational modifications (PTMs). In this review, we discuss the regulatory factors of SERCA2a and provide new insights into future treatments and the direction of heart failure research. In addition, gene therapy for SERCA2a has recently emerged as therapeutic option and hence will be discussed in this review.

Mining for missed sORF-encoded peptides.

INTRODUCTION: Small open reading frames (sORFs) with potential protein-coding capacity have been disclosed in various transcripts, including long noncoding RNAs (LncRNAs), mRNAs (5'-upstream, coding domain, and 3'-downstream), circular RNAs, pri-miRNAs, and ribosomal RNAs (rRNAs). Recent characterization of several sORF-encoded peptides (SEPs or micropeptides) revealed their important roles in many fundamental biological processes in a broad range of species from yeast to human. The success in the mining of micropeptides attributes to the advanced bioinformatics and high-throughput sequencing techniques. Areas covered: sORFs and SEPs were overlooked for their tiny size and the difficulty of identification by bioinformatics analyses. With more and more sORFs and SEPs have been identified, this field has attracted more attention. This review covers recent advances in the strategies for the detection and identification of sORFs and SEPs. Expert commentary: The advantages and drawbacks of the strategies for detection and identification of sORFs and SEPs are discussed, as well as the techniques that are used to decipher the roles of micropeptides in organisms are described.

Viral Short ORFs and Their Possible Functions.

Definition of functional genomic elements is one of the greater challenges of the genomic era. Traditionally, putative short open reading frames (sORFs) coding for less than 100 amino acids were disregarded due to computational and experimental limitations; however, it has become clear over the past several years that translation of sORFs is pervasive and serves diverse functions. The development of ribosome profiling, allowing identification of translated sequences genome wide, revealed wide spread, previously unidentified translation events. New computational methodologies as well as improved mass spectrometry approaches also contributed to the task of annotating translated sORFs in different organisms. Viruses are of special interest due to the selective pressure on their genome size, their rapid and confining evolution, and the potential contribution of novel peptides to the host immune response. Indeed, many functional viral sORFs were characterized to date, and ribosome profiling analyses suggest that this may be the tip of the iceberg. Our computational analyses of sORFs identified by ribosome profiling in DNA viruses demonstrate that they may be enriched in specific features implying that at least some of them are functional. Combination of systematic genome editing strategies with synthetic tagging will take us into the next step-elucidation of the biological relevance and function of this intriguing class of molecules.

Comprehensive Peptide Analysis of Mouse Brain Striatum Identifies Novel sORF-Encoded Polypeptides.

Bio-active peptides are involved in the regulation of most physiological processes in the body. Classical bio-active peptides (CBAPs) are cleaved from a larger precursor protein and stored in secretion vesicles from which they are released in the extracellular space. Recently, another non-classical type of bio-active peptides (NCBAPs) has gained interest. These typically are not secreted but instead appear to be translated from short open reading frames (sORF) and released directly into the cytoplasm. In contrast to CBAPs, these peptides are involved in the regulation of intra-cellular processes such as transcriptional control, calcium handling and DNA repair. However, bio-chemical evidence for the translation of sORFs remains elusive. Comprehensive analysis of sORF-encoded polypeptides (SEPs) is hampered by a number of methodological and biological challenges: the low molecular mass (many 4-10 kDa), the low abundance, transient expression and complications in data analysis. We developed a strategy to address a number of these issues. Our strategy is to exclude false positive identifications. In total sample, we identified 926 peptides originated from 37 known (neuro)peptide precursors in mouse striatum. In addition, four SEPs were identified including NoBody, a SEP that was previously discovered in humans and three novel SEPS from 5' untranslated transcript regions (UTRs).

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

KEY POINTS: Small transmembrane proteins such as FXYDs, which interact with Na+ ,K+ -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. ABSTRACT: Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na+ ,K+ -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

Transcriptome landscape of long intergenic non-coding RNAs in endometrial cancer.

Endometrial cancer is the most common gynecological malignancy in the developed world. It is the fifth most common cancer and accounts for 4.8% of all cancers in women. Long intergenic non-coding RNAs (lincRNAs), a subclass of long non-coding RNAs, are pervasively transcribed throughout the human genome. OBJECTIVE: LincRNA expression patterns in endometrial cancer compared to normal healthy tissue are poorly characterised. In this study, the lincRNA transcriptome of endometrial cancers and adjacent normal endometrium from the same patients was sequenced and compared with transcriptomes of other gynaecologic malignancies including ovarian and cervical cancers. METHODS: RNA was isolated from malignant and adjacent non-affected endometrial tissue from 6 patients with low grade and stage Type I endometrial cancer. Subsequently, Illumina paired-end RNA sequencing was performed, followed by bioinformatics analysis, to determine differential transcriptome expression patterns. RESULTS: LINC00958 was upregulated in all three cancers, and four lincRNAs including LINC01480, LINC00645, LINC00891 and LINC00702 demonstrated exquisite specificity for malignant endometrium compared to normal endometrium while also distinguishing endometrial cancer from ovarian and cervical cancers. Furthermore, LINC01480 has features required to express a micropeptide. CONCLUSIONS: The lincRNAs, characterised in this study, represent high priority genes to be tested for functional significance in the pathogenesis and/or progression of endometrial cancer. Furthermore, lincRNAs have potential to be released into the bloodstream and therefore the four lincRNAs identified here may represent biomarkers for early detection of endometrial cancer without biopsy.