DEGRONOPEDIA: a web server for proteome-wide inspection of degrons.

E3 ubiquitin ligases recognize substrates through their short linear motifs termed degrons. While degron-signaling has been a subject of extensive study, resources for its systematic screening are limited. To bridge this gap, we developed DEGRONOPEDIA, a web server that searches for degrons and maps them to nearby residues that can undergo ubiquitination and disordered regions, which may act as protein unfolding seeds. Along with an evolutionary assessment of degron conservation, the server also reports on post-translational modifications and mutations that may modulate degron availability. Acknowledging the prevalence of degrons at protein termini, DEGRONOPEDIA incorporates machine learning to assess N-/C-terminal stability, supplemented by simulations of proteolysis to identify degrons in newly formed termini. An experimental validation of a predicted C-terminal destabilizing motif, coupled with the confirmation of a post-proteolytic degron in another case, exemplifies its practical application. DEGRONOPEDIA can be freely accessed at degronopedia.com.

Lysine deserts and cullin-RING ligase receptors: Navigating untrodden paths in proteostasis.

The ubiquitin-proteasome system (UPS) governs the degradation of proteins by ubiquitinating their lysine residues. Our study focuses on lysine deserts - regions in proteins conspicuously low in lysine residues - in averting ubiquitin-dependent proteolysis. We spotlight the prevalence of lysine deserts among bacteria leveraging the pupylation-dependent proteasomal degradation, and in the UPS of eukaryotes. To further scrutinize this phenomenon, we focused on human receptors VHL and SOCS1 to ascertain if lysine deserts could limit their ubiquitination within the cullin-RING ligase (CRL) complex. Our data indicate that the wild-type and lysine-free variants of VHL and SOCS1 maintain consistent turnover rates, unaltered by CRL-mediated ubiquitination, hinting at a protective mechanism facilitated by lysine deserts. Nonetheless, we noted their ubiquitination at non-lysine sites, alluding to alternative regulation by the UPS. Our research underscores the role of lysine deserts in limiting CRL-mediated ubiquitin tagging while promoting non-lysine ubiquitination, thereby advancing our understanding of proteostasis.

In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers.

Background: Loss of function mutation in FLG is the major genetic risk factor for atopic dermatitis (AD) and other allergic manifestations. Presently, little is known about the cellular turnover and stability of profilaggrin, the protein encoded by FLG. Since ubiquitination directly regulates the cellular fate of numerous proteins, their degradation and trafficking, this process could influence the concentration of filaggrin in the skin. Objective: To determine the elements mediating the interaction of profilaggrin with the ubiquitin-proteasome system (i.e., degron motifs and ubiquitination sites), the features responsible for its stability, and the effect of nonsense and frameshift mutations on profilaggrin turnover. Methods: The effect of inhibition of proteasome and deubiquitinases on the level and modifications of profilaggrin and processed products was assessed by immunoblotting. Wild-type profilaggrin sequence and its mutated variants were analysed in silico using the DEGRONOPEDIA and Clustal Omega tool. Results: Inhibition of proteasome and deubiquitinases stabilizes profilaggrin and its high molecular weight of presumably ubiquitinated derivatives. In silico analysis of the sequence determined that profilaggrin contains 18 known degron motifs as well as multiple canonical and non-canonical ubiquitination-prone residues. FLG mutations generate products with increased stability scores, altered usage of the ubiquitination marks, and the frequent appearance of novel degrons, including those promoting C-terminus-mediated degradation routes. Conclusion: The proteasome is involved in the turnover of profilaggrin, which contains multiple degrons and ubiquitination-prone residues. FLG mutations alter those key elements, affecting the degradation routes and the mutated products' stability.

Structural interaction fingerprints and machine learning for predicting and explaining binding of small molecule ligands to RNA.

Ribonucleic acids (RNAs) play crucial roles in living organisms and some of them, such as bacterial ribosomes and precursor messenger RNA, are targets of small molecule drugs, whereas others, e.g. bacterial riboswitches or viral RNA motifs are considered as potential therapeutic targets. Thus, the continuous discovery of new functional RNA increases the demand for developing compounds targeting them and for methods for analyzing RNA-small molecule interactions. We recently developed fingeRNAt-a software for detecting non-covalent bonds formed within complexes of nucleic acids with different types of ligands. The program detects several non-covalent interactions and encodes them as structural interaction fingerprint (SIFt). Here, we present the application of SIFts accompanied by machine learning methods for binding prediction of small molecules to RNA. We show that SIFt-based models outperform the classic, general-purpose scoring functions in virtual screening. We also employed Explainable Artificial Intelligence (XAI)-the SHapley Additive exPlanations, Local Interpretable Model-agnostic Explanations and other methods to help understand the decision-making process behind the predictive models. We conducted a case study in which we applied XAI on a predictive model of ligand binding to human immunodeficiency virus type 1 trans-activation response element RNA to distinguish between residues and interaction types important for binding. We also used XAI to indicate whether an interaction has a positive or negative effect on binding prediction and to quantify its impact. Our results obtained using all XAI methods were consistent with the literature data, demonstrating the utility and importance of XAI in medicinal chemistry and bioinformatics.

A novel de novo FEM1C variant is linked to neurodevelopmental disorder with absent speech, pyramidal signs and limb ataxia.

The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e. E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

fingeRNAt-A novel tool for high-throughput analysis of nucleic acid-ligand interactions.

Computational methods play a pivotal role in drug discovery and are widely applied in virtual screening, structure optimization, and compound activity profiling. Over the last decades, almost all the attention in medicinal chemistry has been directed to protein-ligand binding, and computational tools have been created with this target in mind. With novel discoveries of functional RNAs and their possible applications, RNAs have gained considerable attention as potential drug targets. However, the availability of bioinformatics tools for nucleic acids is limited. Here, we introduce fingeRNAt-a software tool for detecting non-covalent interactions formed in complexes of nucleic acids with ligands. The program detects nine types of interactions: (i) hydrogen and (ii) halogen bonds, (iii) cation-anion, (iv) pi-cation, (v) pi-anion, (vi) pi-stacking, (vii) inorganic ion-mediated, (viii) water-mediated, and (ix) lipophilic interactions. However, the scope of detected interactions can be easily expanded using a simple plugin system. In addition, detected interactions can be visualized using the associated PyMOL plugin, which facilitates the analysis of medium-throughput molecular complexes. Interactions are also encoded and stored as a bioinformatics-friendly Structural Interaction Fingerprint (SIFt)-a binary string where the respective bit in the fingerprint is set to 1 if a particular interaction is present and to 0 otherwise. This output format, in turn, enables high-throughput analysis of interaction data using data analysis techniques. We present applications of fingeRNAt-generated interaction fingerprints for visual and computational analysis of RNA-ligand complexes, including analysis of interactions formed in experimentally determined RNA-small molecule ligand complexes deposited in the Protein Data Bank. We propose interaction fingerprint-based similarity as an alternative measure to RMSD to recapitulate complexes with similar interactions but different folding. We present an application of interaction fingerprints for the clustering of molecular complexes. This approach can be used to group ligands that form similar binding networks and thus have similar biological properties. The fingeRNAt software is freely available at https://github.com/n-szulc/fingeRNAt.

The Dose-Dependent Pleiotropic Effects of the UBB+1 Ubiquitin Mutant.

The proteolytic machinery activity diminishes with age, leading to abnormal accumulation of aberrant proteins; furthermore, a decline in protein degradation capacity is associated with multiple age-related proteinopathies. Cellular proteostasis can be maintained via the removal of ubiquitin (Ub)-tagged damaged and redundant proteins by the ubiquitin-proteasome system (UPS). However, during aging, central nervous system (CNS) cells begin to express a frameshift-mutated Ub, UBB+1. Its accumulation is a neuropathological hallmark of tauopathy, including Alzheimer's disease and polyglutamine diseases. Mechanistically, in cell-free and cell-based systems, an increase in the UBB+1 concentration disrupts proteasome processivity, leading to increased aggregation of toxic proteins. On the other hand, a low level of UBB+1 improves stress resistance and extends lifespan. Here we summarize recent findings regarding the impact of UBB+1 on Ub signaling and neurodegeneration. We also review the molecular basis of how UBB+1 affects UPS components as well as its dose-dependent switch between cytoprotective and cytotoxic roles.

BioShell 3.0: Library for Processing Structural Biology Data.

BioShell is an open-source package for processing biological data, particularly focused on structural applications. The package provides parsers, data structures and algorithms for handling and analyzing macromolecular sequences, structures and sequence profiles. The most frequently used routines are accessible by a set of easy-to-use command line utilities for a Linux environment. The full functionality of the package assumes knowledge of C++ or Python to assemble an application using this software library. Since the last publication that announced the version 2.0, the package has been greatly expanded and rewritten in C++ standard 11 (C++11) to improve its modularity and efficiency. A new testing platform has been implemented to continuously test the correctness and integrity of the package. More than two hundred test programs have been published to provide simple examples that can be used as templates. This makes BioShell an easy to use library that greatly speeds up development of bioinformatics applications and web services without compromising computational efficiency.