Search Results (40)

A novel and general approach to the practical ROMP polymerization of cinchona alkaloid derivatives providing novel hybrid materials having quinine attached on a poly(norbornene-5,6-dicarboxyimide) matrix is presented. The concept involves an easy modification of quinine (in general, any cinchona alkaloid) toward clickable 9-azide that reacts with N-propargyl-cis-5-norbornene-exo-2,3-dicarboxylic imide in Cu(I)-catalyzed Huisgen cycloaddition (click chemistry). The resulting monomers undergo a controllable ROMP reaction that leads to novel polymers of a desired length and solubility. This sequence allows for the facile preparation of a regularly decorated polymeric material having one quinine moiety per single mer of the polymer chain inaccessible using typical immobilization methods. A poly(norbornene-5,6-dicarboxyimide) type of polymeric matrix was selected due to the high reactivity of the exo-norbornene motif in Ru(II)-catalyzed ROMP and its chemical and thermal stability as well as convenient, scalable access from inexpensive cis-5-norbornene-exo-2,3-dicarboxylic anhydride (‘one-pot’ Diels–Alder reaction of dicyclopentadiene and maleic anhydride). An appropriate combination of a Grubbs catalyst, Ru(II) (G1, G2), and ROMP conditions allowed for the efficient synthesis of well-defined soluble polymers with mass parameters in the range Mn = 2.24 × 10⁴ – 2.26 × 10⁴ g/mol and Mw = 2.90 × 10⁴–3.05 × 10⁴ g/mol with good polydispersity, Đ_M = 1.32–1.35, and excellent thermal stability (up to 309°C T_d10). Spectroscopic studies (NMR and electronic circular dichroism (ECD)) of these products revealed a linear structure with the slight advantage of a trans-configuration of an olefinic double bond. The resulting short-chain polymer discriminates mandelic acid enantiomers with a preference for the (R)-stereoisomer in spectrofluorimetric assays. This concept seems to be rather general with respect to other molecules dedicated to incorporation into the poly(norbornene-5,6-dicarboxyimide) chain. Full article

Genomic sequences that form three-stranded triplexes (TPXs) under physiological conditions (called T-flipons) play an important role in defining DNA nucleosome-free regions (NFRs). Within these NFRs, other flipon types can cycle conformations to actuate gene expression. The transcripts read from the NFR form condensates that engage proteins and small RNAs. The helicases bound then trigger RNA polymerase release by dissociating the 7SK ribonucleoprotein. The TPXs formed usually incorporate RNA as the third strand. TPXs made only from DNA arise mostly during DNA replication. Many small RNA types (sRNAs) and long noncoding (lncRNA) can direct TPX formation. TPXs made with circular RNAs have greater stability and specificity than those formed with linear RNAs. LncRNAs can affect local gene expression through TPX formation and transcriptional interference. The condensates seeded by lncRNAs are updated by feedback loops involving proteins and noncoding RNAs from the genes they regulate. Some lncRNAs also target distant loci in a sequence-specific manner. Overall, lncRNAs can rapidly evolve by adding or subtracting sequence motifs that modify the condensates they nucleate. LncRNAs show less sequence conservation than protein-coding sequences. TPXs formed by lncRNAs and sRNAs help place nucleosomes to restrict endogenous retroelement (ERE) expression. The silencing of EREs starts early in embryogenesis and is essential for bootstrapping development. Once the system is set, EREs play a different role, with a notable enrichment of Short Interspersed Nuclear Repeats (SINEs) in Enhancer–Promoter condensates. The highly programmable TPX-dependent processes create a chromaverse capable of many complexities. Full article

Viruses manipulate host cellular machinery to propagate their life cycle, with one key strategy being the mimicry of short linear motifs (SLiMs) found in host proteins. While databases continue to expand with virus–host protein–protein interaction (vhPPI) data, accurately predicting viral mimicry remains challenging due to the inherent degeneracy of SLiMs. In this study, we investigate how viral genomic composition influences motif mimicry and the mechanisms through which viruses hijack host cellular functions. We assessed domain–motif interaction (DMI) enrichment differences, and also predicted new DMIs based on known viral motifs with varying stringency levels, using SLiMEnrich v.1.5.1. Our findings reveal that dsDNA viruses capture significantly more known DMIs compared to other viral groups, with dsRNA viruses also exhibiting higher DMI enrichment than ssRNA viruses. Additionally, we identified new vhPPIs mediated via SLiMs, particularly within different viral genomic contexts. Understanding these interactions is vital for elucidating viral strategies to hijack host functions, which could inform the development of targeted antiviral therapies. Full article

OFD1 is a protein involved in many cellular processes, including cilia biogenesis, mitotic spindle assembly, translation, autophagy and the repair of double-strand DNA breaks. Despite many potential interactors identified in high-throughput studies, only a few have been directly confirmed with their binding sites identified. We performed an analysis of the evolutionary conservation of the OFD1 sequence in three clades: 80 Tetrapoda, 144 Vertebrata or 26 Animalia species, and identified 59 protein-binding motifs localized in the OFD1 regions conserved in various clades. Our results indicate that OFD1 contains 14 potential post-translational modification (PTM) sites targeted by at least eight protein kinases, seven motifs bound by proteins recognizing phosphorylated aa residues and a binding site for phosphatase 2A. Moreover, OFD1 harbors both a motif that enables its phosphorylation by mitogen-activated protein kinases (MAPKs) and a specific docking site for these proteins. Generally, our results suggest that OFD1 forms a scaffold for interaction with many proteins and is tightly regulated by PTMs and ligands. Future research on OFD1 should focus on the regulation of OFD1 function and localization. Full article

Effectors are key organism-associated molecules that aid in the establishment of interactions with other organisms. Effectoromics has become an important area of research in phytopathology. The lack of sequence conservation among effectors, even in closely related organisms, has led us to believe that effectors from organisms of different kingdoms are completely unrelated, which has fostered the independent development of effector identification strategies in bacteria, fungi, phytoplasmas, etc. This review focuses on the different algorithms available for effector identification in different plant pathogens and pests, using the following classification: (1) translocated effectors (bacteria, oomycete) and (2) secreted effectors (phytoplasmas, fungi, insects, nematodes). The objective of this type of classification is to identify, for the first time, the common features that exist among these organisms to streamline future effectoromics identification strategies. Among the organisms’ commonalities, certain bacteria, fungi, phytoplasmas, and nematodes may cause similar symptoms, and some of their effectors may target the same proteins or biological processes in the plant hosts. The integration of effector analyses of organisms of different living kingdoms, through the identification of common short linear motifs, domains, and three-dimensional structures, may aid in the development of novel algorithms for effectoromics. Future algorithms may contemplate these highlighted features and will be better equipped to identify not only canonical effectors but highly elusive non-canonical effectors as well. Full article

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-causing virus that establishes life-long infection. KSHV is implicated in the etiology of Kaposi’s sarcoma, and a number of rare hematopoietic malignancies. The present study focuses on the KSHV open reading frame 20 (ORF20), a member of the conserved herpesvirus UL24 protein family containing five conserved homology domains and a conserved PD-(D/E)XK putative endonuclease motif, whose nuclease function has not been established to date. ORF20 encodes three co-linear protein isoforms, full length, intermediate, and short, though their differential functions are unknown. In an effort to determine the role of ORF20 during KSHV infection, we generated a recombinant ORF20-Null KSHV genome, which fails to express all three ORF20 isoforms. This genome was reconstituted in iSLK cells to establish a latent infection, which resulted in an accelerated transcription of viral mRNAs, an earlier accumulation of viral lytic proteins, an increase in the quantity of viral DNA copies, and a significant decrease in viral yield upon lytic reactivation. This was accompanied by early cell death of cells infected with the ORF20-Null virus. Functional complementation of the ORF20-Null mutant with the short ORF20 isoform rescued KSHV production, whereas its endonuclease mutant form failed to enhance lytic reactivation. Complementation with the short isoform further revealed a decrease in cell death as compared with ORF20-Null virus. Finally, expression of IL6 and CXCL8, previously shown to be affected by the hCMV UL24 homolog, was relatively low upon reactivation of cells infected with the ORF20-Null virus. These findings suggest that ORF20 protein, with its putative endonuclease motif, promotes coordinated lytic reactivation for increased infectious particle production. Full article

The involvement of carbonic anhydrases (CAs) in a myriad of biological events makes the development of new inhibitors of these metalloenzymes a hot topic in current Medicinal Chemistry. In particular, CA IX and XII are membrane-bound enzymes, responsible for tumour survival and chemoresistance. Herein, a bicyclic carbohydrate-based hydrophilic tail (imidazolidine-2-thione) has been appended to a CA-targeting pharmacophore (arylsulfonamide, coumarin) with the aim of studying the influence of the conformational restriction of the tail on the CA inhibition. For this purpose, the coupling of sulfonamido- or coumarin-based isothiocyanates with reducing 2-aminosugars, followed by the sequential acid-promoted intramolecular cyclization of the corresponding thiourea and dehydration reactions, afforded the corresponding bicyclic imidazoline-2-thiones in good overall yield. The effects of the carbohydrate configuration, the position of the sulfonamido motif on the aryl fragment, and the tether length and substitution pattern on the coumarin were analysed in the in vitro inhibition of human CAs. Regarding sulfonamido-based inhibitors, the best template turned out to be a d-galacto-configured carbohydrate residue, meta-substitution on the aryl moiety (9b), with K_i against CA XII within the low nM range (5.1 nM), and remarkable selectivity indexes (1531 for CA I and 181.9 for CA II); this provided an enhanced profile in terms of potency and selectivity compared to more flexible linear thioureas 1–4 and the drug acetazolamide (AAZ), used herein as a reference compound. For coumarins, the strongest activities were found for substituents devoid of steric hindrance (Me, Cl), and short linkages; derivatives 24h and 24a were found to be the most potent inhibitors against CA IX and XII, respectively (K_i = 6.8, 10.1 nM), and also endowed with outstanding selectivity (K_i > 100 µM against CA I, II, as off-target enzymes). Docking simulations were conducted on 9b and 24h to gain more insight into the key inhibitor–enzyme interactions. Full article

All proteins have a carboxyl terminus, and we previously summarized eight mutations in binding and trafficking sequence determinants in the C-terminus that, when disrupted, cause human diseases. These sequence elements for binding and trafficking sites, as well as post-translational modifications (PTMs), are called minimotifs or short linear motifs. We wanted to determine how frequently mutations in minimotifs in the C-terminus cause disease. We searched specifically for PTMs because mutation of a modified amino acid almost always changes the chemistry of the side chain and can be interpreted as loss-of-function. We analyzed data from ClinVar for disease variants, Minimotif Miner and the C-terminome for PTMs, and RefSeq for protein sequences, yielding 20 such potential disease-causing variants. After additional screening, they include six with a previously reported PTM disruption mechanism and nine with new hypotheses for mutated minimotifs in C-termini that may cause disease. These mutations were generally for different genes, with four different PTM types and several different diseases. Our study helps to identify new molecular mechanisms for nine separate variants that cause disease, and this type of analysis could be extended as databases grow and to binding and trafficking motifs. We conclude that mutated motifs in C-termini are an infrequent cause of disease. Full article

The PDZ domains are modular domains that recognise short linear C-terminal sequences in proteins that organise the formation of complex multi-component assemblies. We have crystallised the third PDZ domain of the neuronal postsynaptic density-95 protein (PSD95-PDZ3) at mildly acidic pH conditions and obtained up to four polymorphs. Thus, below pH 4.0, the protein crystallised into prism-shaped crystals that belonged to the trigonal space group P3₁12. In contrast, above this pH value, the crystals’ shape changes to long needles belonging to the monoclinic P2₁ and two different orthorhombic packings of the P2₁2₁2₁ space group. In addition, all the polymorphs share the main crystallographic interface, where the sidechain of the Asp332 imitates the binding of the C-terminal moiety to the canonical binding motif. Furthermore, we have analysed how changes in the ionisation state of some specific residues might be critical for crystallising the different polymorphs. The analysis of these polymorphs provides clues on the relevance of specific protein-protein interactions in protein crystallisation. However, these structures allow dissecting those electrostatic interactions that play a role in the conformation adopted by some residues and the extra-domain components upon binding C-terminal sequences. Full article

Protein arginine methylation is an extensive and functionally significant post-translational modification. However, little is known about its role in differentiation at the systems level. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics of whole proteome analysis in proliferating or five-day differentiated mouse C2C12 myoblasts, followed by high-resolution mass spectrometry, biochemical assays, and specific immunoprecipitation of mono- or dimethylated arginine peptides, we identified several protein families that were differentially methylated on arginine. Our study is the first to reveal global changes in the arginine mono- or dimethylation of proteins in proliferating myoblasts and differentiated myocytes and to identify enriched protein domains and novel short linear motifs (SLiMs). Our data may be crucial for dissecting the links between differentiation and cancer growth. Full article

Short linear motifs (SLiMs) are evolutionarily conserved functional modules of proteins composed of 3 to 10 residues and involved in multiple cellular functions. Here, we performed a search for SLiMs that exert sequence similarity to two segments of alpha-fetoprotein (AFP), a major mammalian embryonic and cancer-associated protein. Biological activities of the peptides, LDSYQCT (AFP_14–20) and EMTPVNPGV (GIP-9), have been previously confirmed under in vitro and in vivo conditions. In our study, we retrieved a vast array of proteins that contain SLiMs of interest from both prokaryotic and eukaryotic species, including viruses, bacteria, archaea, invertebrates, and vertebrates. Comprehensive Gene Ontology enrichment analysis showed that proteins from multiple functional classes, including enzymes, transcription factors, as well as those involved in signaling, cell cycle, and quality control, and ribosomal proteins were implicated in cellular adaptation to environmental stress conditions. These include response to oxidative and metabolic stress, hypoxia, DNA and RNA damage, protein degradation, as well as antimicrobial, antiviral, and immune response. Thus, our data enabled insights into the common functions of SLiMs evolutionary conserved across all taxonomic categories. These SLiMs can serve as important players in cellular adaptation to stress, which is crucial for cell functioning. Full article

Colorectal tumorigenesis is driven by alterations in genes and proteins responsible for cancer initiation, progression, and invasion. This multistage process is based on a dense network of protein–protein interactions (PPIs) that become dysregulated as a result of changes in various cell signaling effectors. PPIs in signaling and regulatory networks are known to be mediated by short linear motifs (SLiMs), which are conserved contiguous regions of 3–10 amino acids within interacting protein domains. SLiMs are the minimum sequences required for modulating cellular PPI networks. Thus, several in silico approaches have been developed to predict and analyze SLiM-mediated PPIs. In this review, we focus on emerging evidence supporting a crucial role for SLiMs in driver pathways that are disrupted in colorectal cancer (CRC) tumorigenesis and related PPI network alterations. As a result, SLiMs, along with short peptides, are attracting the interest of researchers to devise small molecules amenable to be used as novel anti-CRC targeted therapies. Overall, the characterization of SLiMs mediating crucial PPIs in CRC may foster the development of more specific combined pharmacological approaches. Full article

Intrinsically disordered regions (IDRs) in protein sequences are flexible, have low structural constraints and as a result have faster rates of evolution. This lack of evolutionary conservation greatly limits the use of sequence homology for the classification and functional assessment of IDRs, as opposed to globular domains. The study of IDRs requires other properties for their classification and functional prediction. While composition bias is not a necessary property of IDRs, compositionally biased regions (CBRs) have been noted as frequent part of IDRs. We hypothesized that to characterize IDRs, it could be helpful to study their overlap with particular types of CBRs. Here, we evaluate this overlap in the human proteome. A total of 2/3 of residues in IDRs overlap CBRs. Considering CBRs enriched in one type of amino acid, we can distinguish CBRs that tend to be fully included within long IDRs (R, H, N, D, P, G), from those that partially overlap shorter IDRs (S, E, K, T), and others that tend to overlap IDR terminals (Q, A). CBRs overlap more often IDRs in nuclear proteins and in proteins involved in liquid-liquid phase separation (LLPS). Study of protein interaction networks reveals the enrichment of CBRs in IDRs by tandem repetition of short linear motifs (rich in S or P), and the existence of E-rich polar regions that could support specific protein interactions with non-specific interactions. Our results open ways to pin down the function of IDRs from their partial compositional biases. Full article

Short linear motifs (SLiMs) are short linear sequences that can mediate protein–protein interaction. Mimicking eukaryotic SLiMs to compete with extra- or intracellular binding partners, or to sequester host proteins is the crucial strategy of viruses to pervert the host system. Evolved proteins in viruses facilitate minimal protein–protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about SARS-CoV-2 SLiMs is known, especially across SARS-CoV-2 variants. Through the ELM database-based sequence analysis of spike proteins from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant, namely, LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants. Full article

Short linear motifs (SLiMs) are evolutionarily conserved functional modules of proteins that represent amino acid stretches composed of 3 to 10 residues. The biological activities of two short peptide segments of human alpha-fetoprotein (AFP), a major embryo-specific and cancer-related protein, have been confirmed experimentally. This is a heptapeptide segment LDSYQCT in domain I designated as AFP_14–20 and a nonapeptide segment EMTPVNPGV in domain III designated as GIP-9. In our work, we searched the UniprotKB database for human proteins that contain SLiMs with sequence similarity to the both segments of human AFP and undertook gene ontology (GO)-based functional categorization of retrieved proteins. Gene set enrichment analysis included GO terms for biological process, molecular function, metabolic pathway, KEGG pathway, and protein–protein interaction (PPI) categories. We identified the SLiMs of interest in a variety of non-homologous proteins involved in multiple cellular processes underlying embryonic development, cancer progression, and, unexpectedly, the regulation of redox homeostasis. These included transcription factors, cell adhesion proteins, ubiquitin-activating and conjugating enzymes, cell signaling proteins, and oxidoreductase enzymes. They function by regulating cell proliferation and differentiation, cell cycle, DNA replication/repair/recombination, metabolism, immune/inflammatory response, and apoptosis. In addition to the retrieved genes, new interacting genes were identified. Our data support the hypothesis that conserved SLiMs are incorporated into non-homologous proteins to serve as functional blocks for their orchestrated functioning. Full article