Search Results (153)

MuRF1 [muscle RING (Really Interesting New Gene)-finger protein-1] is an ubiquitin-protein ligase (E3), which encode by TRIM63 (tripartite motif containing 63) gene, playing a crucial role in regulating cardiac muscle size and function through ubiquitylation. Among hypertrophic cardiomyopathy (HCM) patients, 24 TRIM63 variants have been identified, with 1 additional variant linked to restrictive cardiomyopathy. However, only three variants have been previously investigated for their functional effects. The structural impacts of the 25 variants remain unexplored. This study investigated the effects of 25 MuRF1 variants on ubiquitylation activity using in vitro ubiquitylation assays and structural predictions using computational approaches. The variants were generated using site-directed PCR (Polymerase Chain Reaction) mutagenesis and subsequently purified with amylose affinity chromatography. In vitro ubiquitylation assays demonstrated that all 25 variants compromised the ability of MuRF1 to monoubiquitylate a titin fragment (A168-A170), while 17 variants significantly impaired or completely abolished auto-monoubiquitylation. Structural modelling predicted that 10 MuRF1 variants disrupted zinc binding or key stabilising interactions, compromising structural integrity. In contrast, three variants were predicted to enhance the structural stability of MuRF1, while six others were predicted to have no discernible impact on the structure. This study underscores the importance of functional assays and structural predictions in evaluating MuRF1 variant pathogenicity and provides novel insights into mechanisms by which these variants contribute to HCM and related cardiomyopathies. Full article

The cell wall integrity (CWI) pathway is responsible for transcriptional regulation of cell wall remodeling in response to cell wall stress. How cell wall remodeling mediated by the CWI pathway is effected by inputs from other signaling pathways is not well understood. Here, we demonstrate that the Mck1 kinase cooperates with Slt2, the MAP kinase of the CWI pathway, to promote cell wall thickening in glucose-starved cells. Integrative analyses of the transcriptome, proteome and metabolic profiling indicate that Mck1 is required for the accumulation of UDP-glucose (UDPG), the substrate for β-glucan synthesis, through the activation of two regulons: the Msn2/4-dependent stress response and the Cat8-/Adr1-mediated metabolic reprogram dependent on the SNF1 complex. Analysis of the phosphoproteome suggests that similar to mammalian Gsk-3 kinases, Mck1 is involved in the regulation of cytoskeleton-dependent cellular processes, metabolism, signaling and transcription. Specifically, Mck1 may be implicated in the Snf1-dependent metabolic reprogram through PKA inhibition and SAGA (Spt-Ada-Gcn5 acetyltransferase)-mediated transcription activation, a hypothesis further underscored by the significant overlap between the Mck1- and Gcn5-activated transcriptomes. Phenotypic analysis also supports the roles of Mck1 in actin cytoskeleton-mediated exocytosis to ensure plasma membrane homeostasis and cell wall remodeling in cell wall-stressed cells. Together, these findings not only reveal the novel functions of Mck1 in metabolic reprogramming and polarized growth but also provide valuable omics resources for future studies to uncover the underlying mechanisms of Mck1 and other Gsk-3 kinases in cell growth and stress response. Full article

Ubiquitylation is a post-translational modification originally identified as the first step in protein degradation by the ubiquitin–proteasome system. Ubiquitylation is also known to regulate many cellular processes without degrading the ubiquitylated proteins. Substrate proteins are specifically recognized and ubiquitylated by ubiquitin ligases. It is necessary to identify the substrates for each ubiquitin ligase to understand the physiological and pathological roles of ubiquitylation. Recently, a promiscuous mutant of a biotin ligase derived from Escherichia coli, BioID, and its variants have been utilized to analyze protein–protein interaction. In this review, we summarize the current knowledge regarding the molecular mechanisms underlying ubiquitylation, BioID-based approaches for interactome studies, and the application of BirA and its variants for the identification of ubiquitin ligase substrates. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver disorder with limited treatment options. This review explores the role of post-translational modifications (PTMs) in MASLD pathogenesis, highlighting their potential as therapeutic targets. We discuss the impact of PTMs, including their phosphorylation, ubiquitylation, acetylation, and glycosylation, on key proteins involved in MASLD, drawing on studies that use both human subjects and animal models. These modifications influence various cellular processes, such as lipid metabolism, inflammation, and fibrosis, contributing to disease progression. Understanding the intricate PTM network in MASLD offers the potential for developing novel therapeutic strategies that target specific PTMs to modulate protein function and alleviate disease pathology. Further research is needed to fully elucidate the complexity of PTMs in MASLD and translate these findings into effective clinical applications. Full article

Reporter systems are widely used to study biomolecular interactions and processes in vivo, representing one of the basic tools used to characterize synthetic regulatory circuits. Here, we developed a method that enables the monitoring of RNA–protein interactions through a reporter system in bacteria with high temporal resolution. For this, we used a Real-Time Protein Expression Assay (RT-PEA) technology for real-time monitoring of a fluorescent reporter protein, while having bacteria growing on solid media. Experimental results were analyzed by fitting a three-variable Gompertz growth model. To validate the method, the interactions between a set of RNA sequences and the RNA-binding protein (RBP) Musashi-1 (MSI1) were evaluated, as well as the allosteric modulation of the interaction by a small molecule (oleic acid). This new approach proved to be suitable to quantitatively characterize RNA–RBP interactions, thereby expanding the toolbox to study molecular interactions in living bacteria, including allosteric modulation, with special relevance for systems that are not suitable to be studied in liquid media. Full article

Recent discoveries revealed mechanistic insights into the control of adipogenesis by the Constitutive Photomorphogenesis 9 Signalosome (CSN) and its variants, CSN^CSN7A and CSN^CSN7B, which differ in the paralog subunits, CSN7A and CSN7B. CSN^CSN7A and CSN^CSN7B variants form permanent complexes with cullin-RING-ubiquitin ligases 3 and 4A (CRL3 and CRL4A), respectively. These complexes can be found in most eukaryotic cells and represent a critical reservoir for cellular functions. In an early stage of adipogenesis, mitotic clonal expansion (MCE), CSN-CRL1, and CSN^CSN7B-CRL4A are blocked to ubiquitinate the cell cycle inhibitor p27^KIP, leading to cell cycle arrest. In addition, in MCE CSN-CRL complexes rearrange the cytoskeleton for adipogenic differentiation and CRL3^KEAP1 ubiquitylates the inhibitor of adipogenesis C/EBP homologous protein (CHOP) for degradation by the 26S proteasome, an adipogenesis-specific proteolysis. During terminal adipocyte differentiation, the CSN^CSN7A-CRL3 complex is recruited to a lipid droplet (LD) membrane by RAB18. Currently, the configuration of the substrate receptors of CSN^CSN7A-CRL3 on LDs is unclear. CSN^CSN7A-CRL3 is activated by neddylation on the LD membrane, an essential adipogenic step. Damage to CSN/CUL3/CUL4A genes is associated with diverse diseases, including obesity. Due to the tremendous impact of CSN-CRLs on adipogenesis, we need strategies for adequate treatment in the event of malfunctions. Full article

Background/Objectives: Lung cancer ranks as the leading cause of cancer-related deaths globally and is highly associated with cisplatin resistance due to both intrinsic and extrinsic mechanisms. Proliferating Cell Nuclear Antigen (PCNA) plays a critical role in molecular processes, such as DNA replication and repair, chromatin structure maintenance, and cell cycle progression. PCNA is known as a molecular marker for proliferation and an excellent inhibition target to shut down highly proliferative cells. One of the mechanisms of cisplatin resistance is the increase in DNA repair, and studies have reported an association between PCNA, lung cancer, and cisplatin treatment. The present study aimed to characterize the absence of PCNA in A549 lung adenocarcinoma cells. Methods: Employing a CRISPR/Cas9 gene-editing approach, we generated a monoclonal cell culture, termed PKO (PCNA knockout). Results: PKO cells exhibited a residual PCNA expression, significantly decreased clonogenic potential and ubiquitylation at K164 residue. IC50 assay suggested that PKO cells could not acquire cisplatin resistance when compared to PX. After cisplatin treatment, PKO cells presented impaired ubiquitylation and did not have increased STAT3 phosphorylation (Tyr705), a previously characterized mechanism of cisplatin resistance. Conclusions: We suggest that PCNA participates in cisplatin resistance in A549, partially by DNA damage tolerance through failure on PCNA monoubiquitylation, and its inhibition may be an approach to circumvent cisplatin resistance. Full article

Ubiquitylation is a post-translational modification that modulates protein function and stability. It is orchestrated by the concerted action of three types of enzymes, with substrate specificity governed by ubiquitin ligases (E3s), which may exist as single proteins or as part of multi-protein complexes. Although Cullin (CUL) proteins lack intrinsic enzymatic activity, they participate in the formation of active ubiquitin ligase complexes, known as Cullin-Ring ubiquitin Ligases (CRLs), through their association with ROC1 or ROC2, along with substrate adaptor and receptor proteins. Mammalian genomes encode several CUL proteins (CUL1–9), each contributing to distinct CRLs. Among these CUL proteins, CUL1, CUL3, and CUL4 are believed to be the most ancient and evolutionarily conserved from yeast to mammals, with CUL4 uniquely duplicated in vertebrates. Genetic evidence strongly implicates CUL4-based ubiquitin ligases (CRL4s) in chromatin regulation across various species and suggests that, in vertebrates, CRL4s have also acquired a cytosolic role, which is facilitated by a cytosol-localizing paralog of CUL4. Substrates identified through biochemical studies have elucidated the molecular mechanisms by which CRL4s regulate chromatin and cytosolic processes. The substantial body of knowledge on CUL4 biology amassed over the past two decades provides a unique opportunity to explore the functional evolution of CRL4. In this review, we synthesize the available structural, genetic, and biochemical data on CRL4 from various model organisms and discuss the conserved and novel functions of CRL4s. Full article

Tripartite motif (TRIM) family proteins, distinguished by their N-terminal region that includes a Really Interesting New Gene (RING) domain with E3 ligase activity, two B-box domains, and a coiled-coil region, have been recognized as significant contributors in carcinogenesis, primarily via the ubiquitin–proteasome system (UPS) for degrading proteins. Mechanistically, these proteins modulate a variety of signaling pathways, including Wnt/β-catenin, PI3K/AKT, and TGF-β/Smad, contributing to cellular regulation, and also impact cellular activities through non-signaling mechanisms, including modulation of gene transcription, protein degradation, and stability via protein–protein interactions. Currently, growing evidence indicates that TRIM proteins emerge as potential regulators in gastric cancer, exhibiting both tumor-suppressive and oncogenic roles. Given their critical involvement in cellular processes and the notable challenges of gastric cancer, exploring the specific contributions of TRIM proteins to this disease is necessary. Consequently, this review elucidates the roles and mechanisms of TRIM proteins in gastric cancer, emphasizing their potential as therapeutic targets and prognostic factors. Full article

Cryptic transcription refers to the unintended expression of non-canonical sites within the genome, producing aberrant RNA and proteins that may disrupt cellular functions. In this opinion piece, I will explore the role of histone modifications in modulating cryptic transcription and its implications for gene expression and cellular integrity, particularly with a focus on H3K36 and H3K4 methylation marks. H3K36 tri-methylation plays a crucial role in maintaining chromatin integrity by facilitating the recruitment of the Rpd3S histone deacetylase (HDAC) complex, which helps restore closed chromatin states following transcription and prevents cryptic initiation within gene bodies. In parallel, crosstalk between H3K4 di-methylation and histone ubiquitylation and sumoylation is critical for recruiting the Set3 HDAC complex, which maintains low histone acetylation levels in gene bodies and further suppresses cryptic transcription. Therefore, by elucidating these regulatory mechanisms, this opinion highlights the intricate interplay of histone modifications in preserving transcriptional fidelity and suggests potential pathways for future research to develop novel therapies for age-related disorders and other diseases associated with dysregulated gene expression. Full article

Activation of the ubiquitin ligase APC/C by the protein Cdc20 is an essential requirement for proper cell division in higher organisms, including humans. APC/C is the ultimate effector of the Spindle Assembly Checkpoint (SAC), the signalling system that monitors the proper attachment of chromosomes to microtubules during cell division. Defects in this process result in genome instability and cancer. Interfering with APC/C substrate ubiquitylation in cancer cells delays mitotic exit, which induces cell death. Therefore, impairing APC/C function represents an opportunity for the treatment of cancer and malignancies associated with SAC dysregulation. In this study, we report a new class of pyrimidinethylcarbamate apcin analogues that interfere with APC/C activity in 2D and 3D breast cancer cells. The new pyrimidinethylcarbamate apcin analogues exhibited higher cytotoxicity than apcin in all breast cancer cell subtypes investigated, with much lower cytotoxicity observed in fibroblasts and RPE-1 cells. Further molecular rationalisation of apcin and its derivatives was conducted using molecular docking studies. These structural modifications selected from the in silico studies provide a rational basis for the development of more potent chemotypes to treat highly aggressive breast cancer and possibly other aggressive tumour types of diverse tissue origins. Full article

The Ubiquitin–Proteasome System (UPS) governs numerous cellular processes by modulating protein stability and activity via the conjugation of the small protein ubiquitin, either as a single molecule or as linkages with distinct functions. Dysregulation of the UPS has been associated with many diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Ubiquitin-conjugating enzymes (E2s) are important players of the UPS that work together with ubiquitin ligases (E3s) to promote substrate ubiquitylation. In this study, we conduct a comparative proteome-wide abundance profiling of S. cerevisiae cells during the exponential growth phase with and without heat shock treatment. We focus on cells with deletions of the two highly homologous E2s, UBC4 or UBC5, and use isobaric tag-based quantitative mass spectrometry to elucidate differences and similarities in their proteomic profiles. Our analysis revealed that the deletion of Ubc4 has a stronger effect on the proteome compared to the deletion of Ubc5, particularly in exponentially growing cells. In contrast, the effect on the proteome of deleting Ubc5 becomes evident only after heat shock, and even then, it remains minor compared to Ubc4. Furthermore, we identified proteins increasing in the absence of each enzyme, which may represent candidate substrates, potentially contributing to a better understanding of their cellular role. Full article

The yeast ATPase Cdc48 (known as p97/VCP in human cells) plays an important role in the Ubiquitin Proteasome System. VCP is essential for cancer cell proliferation, and its dysregulation has been implicated in several neurodegenerative diseases. Cdc48 functions by extracting ubiquitylated proteins from membranes, protein complexes and chromatin by often facilitating their proteasomal degradation. Specific adaptors or cofactors, primarily belonging to the UBX domain-containing protein family (which has seven members in Saccharomyces cerevisiae) recruit Cdc48 to ubiquitylated proteins. Here, we employed sample multiplexing-based quantitative mass spectrometry to profile global protein abundance in p97 adaptor deletion strains, specifically comparing seven single deletion strains of UBX domain-containing proteins and the Cuz1 deletion strain, which belongs to the zinc finger AN1-type domain protein family. We observed that each strain showed unique sets of differentially abundant proteins compared to the wild type. Our analysis also revealed a role for Ubx3 in maintaining wild type levels of mitochondrial proteins. Overall, we identified ~1400 differentially abundant proteins in the absence of a specific Cdc48 adaptor. This unique dataset offers a valuable resource for studying the functions of these adaptors, aiming to achieve a better understanding of the cellular processes regulated by Cdc48 itself and to deepen our understanding of the Ubiquitin Proteasome System. Full article

Ubiquitination is one of the most important post-translational modifications in eukaryotes. The ubiquitination cascade includes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The E3 ligases, responsible for substrate recognition, are the most abundant and varied proteins in the cascade and the most studied. SKP1-CUL1-F-Box (SCF)-type E3 ubiquitin ligases are multi-subunit RING (Really Interesting New Gene) E3 ubiquitin ligases, composed of CUL1 (Cullin 1), RBX1 (RING BOX 1), SKP1 (S-phase Kinase-associated Protein 1), and F-box proteins. In vitro ubiquitination assays, used for studying the specific recognition of substrate proteins by E3 ubiquitin ligases, require the purification of all components involved in the cascade, and for assays with SCF-type E3 ligases, additional proteins (several SCF complex subunits). Here, the Duet expression system was used to co-express E1, E2, ubiquitin, ubiquitylation target (substrate), and the four subunits of a SCF-type E3 ligase in E. coli. When these proteins co-exist in bacterial cells, ubiquitination occurs and can be detected by Western Blot. The effectiveness of this bacterial system for detecting ubiquitination cascade activity was demonstrated by replicating both AtSCF^TIR1-mediated and human SCF^FBXO28-mediated ubiquitylation in bacteria. This system provides a basic but adaptable platform for the study of SCF-type E3 ubiquitin ligases. Full article