Search Results (163)

C. burnetii (Cb) is an obligate intracellular bacterial pathogen that replicates within alveolar macrophages following aerosol infection. Unlike most intracellular bacteria, Cb establishes a lysosome-derived replicative niche (Coxiella-containing vacuole or CCV) through the action of its Type IVB secretion system (T4BSS). This system translocates a large repertoire of effector proteins into the host cytoplasm after phagosome acidification. These effectors interfere with diverse signaling pathways to co-opt host processes, such as vesicle trafficking, ubiquitylation, gene expression and lipid metabolism, promoting pathogen survival without triggering robust proinflammatory signaling or host cell death pathways. This effector-triggered immune silencing is particularly unique given the central role of macrophages as innate immune sentinels. In this review, we examine Cb T4BSS effectors that have been characterized as central determinants of innate immunity modulation. We discuss innate immune sensing pathways potentially engaged during infection, including Toll-like receptors, NOD-like receptors, RIG-I-like receptors, inflammasomes, and interferon signaling pathways, and highlight evidence indicating that these pathways are actively suppressed. Emphasis is placed on effector-mediated regulation of NF-κB signaling, type I interferon responses, and inflammasome activation. Finally, we address unresolved questions related to effector-triggered immunity, redundancy in immune suppression, and discrepancies between in vitro and in vivo infection models. Full article

Multiple viruses suppress the antiviral defense system of the host for optimal growth and pathogenesis by co-opting the ubiquitin-mediated proteasomal system (UPS) that promotes the degradation of cellular substrates belonging to the interferon pathway. In the Orthopneumovirus genus, respiratory syncytial virus (RSV), a significant pathogen in human and other animals, employs a pair of viral nonstructural proteins (NS1, NS2) to assemble the UPS. The lack of experimental three-dimensional structures of the substrate proteins and the NS-assembled UPS has impeded progress in our understanding of the mechanism of this assembly process. In an effort to remedy this deficiency, I have taken advantage of the burgeoning field of AI (artificial intelligence) and machine learning programs, such as AlphaFold3, to model the pre-ubiquitylation cores in various combination of the subunits to construct three-dimensional structures, named ‘computed structure models’ (CSMs). The UPS core universally comprises an adapter protein connected to the “substrate” that is to be degraded by the “substrate receptor”. The NS proteins are believed to act as receptors, and cellular Elongin BC as an adapter. These CSMs lend support to the biochemical results where known while also suggesting that the complete core of three proteins is energetically more stable than a complex of only the NS protein and the substrate. In the absence of experimental structures, these results offer, for the first time, a mechanistic insight into RSV-triggered assembly of the UPS, which should allow for a better design of future experiments, and eventually new antiviral regimens. Full article

Tankyrases (TNKS1 and TNKS2) are multifunctional enzymes of the poly(ADP-ribose) polymerase (PARP) family that regulate cellular homeostasis by catalyzing poly(ADP-ribosyl)ation and stabilizing protein–protein interactions through their ankyrin repeat clusters. By engaging with diverse sets of proteins, TNKSs act as central hubs that coordinate signaling and metabolic pathways. In this review, we discuss how TNKS –protein interactions underpin their roles across multiple biological pathways, including Wnt/β-catenin, YAP and SRC signaling, mTORC1 signaling, DNA damage repair (via PARP crosstalk and recruitment of repair factors), telomere maintenance, cell-cycle regulation, glucose metabolism, cytoskeleton rearrangement, autophagy, proteasomal degradation, and apoptosis. We highlight the structural basis of these interactions, emphasizing ankyrin repeat domain recognition motifs and the consequences of TNKS-mediated PARylation on protein stability and localization. By integrating findings from oncology, virology, and metabolism, we illustrate how TNKS functions as a nodal regulator linking genome stability, signaling fidelity, and metabolic control. The interplay between TNKS and these varied pathways is essential for the well-being of the organism, with its dysregulation having severe biological and clinical consequences, which are discussed here. Finally, we consider therapeutic implications of disrupting TNKS–protein interactions, with particular attention paid to selective small-molecule inhibitors and their translational potential in cancer, viral infections, and degenerative diseases. Full article

In Mammalia, the COP9 signalosome (CSN) is associated with cullin-RING ubiquitin ligases (CRLs). This study focuses on the variants CSNCSN7A and CSNCSN7B, which form complexes with CRL3 and CRL4A, respectively. Although some research has been conducted on the assembly of the complexes, little is known about their breakdown. Here, we show that entire CSNCSN7A-CRL3 and CSNCSN7B-CRL4A complexes are degraded via autophagy. CSN-CRL complexes are degraded in the absence of serum via bulk autophagy and in the presence of the specific inhibitor of CSN, CSN5i-3, via selective macroautophagy. Surprisingly, the self-ubiquitylation of cullins in the CRLs was identified as a specific signal for selective macroautophagy. The self-ubiquitylation of cullins takes place in the presence of CSN5i-3, and CSN-CRL complexes are expelled from the nucleus to be degraded in the cytosol. Selective macroautophagy can be blocked by chloroquine, a specific inhibitor of autophagy. Interestingly, the process can also be inhibited by MLN4924, a neddylation inhibitor. Confocal fluorescence microscopy illustrates the interaction of CSN subunits with ATG8, as well as with RAB7, both in HeLa and in LiSa-2 cells. Confocal fluorescence microscopy produces images that suggest the localization of CSN-CRL particles in autophagosomes. Our data place CSN-CRL in the category of large complexes that are degraded through autophagy. Full article

Chlamydia trachomatis has a significant impact on public health, especially among adolescents and young women; it primarily affects urogenital epithelial cells, leading to cervicitis and urethritis, with >90% of cases showing no symptoms. Consequently, chlamydial infections are commonly misdiagnosed, and, if untreated, they may result in severe reproductive sequelae including infertility. A better understanding of C. trachomatis cell biology and bacterial–host cell interactions may be helpful to identify strategies able to counter its transmission among the population, as well as its dissemination in reproductive tissues, reducing the risk of developing severe reproductive sequelae. Therefore, the present review aims to summarize the evidence on the interplay between C. trachomatis and the host defence factors within the cervicovaginal environment. The sophisticated strategies employed by this clinically significant pathogen to counteract these mechanisms are also discussed. In the literature, the main defence factors include the microbiota dominated by Lactobacillus crispatus and several molecules like lactoferrin, able to protect the cervicovaginal microenvironment against C. trachomatis through several mechanisms (e.g., EB coaggregation and competitive exclusion, as well as anti-inflammatory activity). However, the major player in clearing chlamydial infections remains the interferon-gamma (IFN-γ) produced by natural killer and T cells, via the depletion of critical nutrients for C. trachomatis such as tryptophan, or via the ubiquitylation and destruction of chlamydial inclusions. Full article

The dysregulation of CK1 isoforms is linked to various types of diseases, including neurodegeneration and different types of neoplasia such as colon, pancreatic, breast, and ovarian cancer. For CK1 isoforms, a plethora of effective small molecule inhibitors are available. However, only a few degraders of CK1α and, more recently, proteolysis targeting chimeras (PROTACs) for CK1δ/CK1ε have been reported. In this study, we applied the PROTAC concept by harnessing molecular modelling to design and synthesize a series of candidate CK1δ-targeting PROTACs based on a highly specific and potent benzothiazole-based CK1δ inhibitor that we previously developed in our lab. In the present study, we established a modular synthetic platform to systematically generate a set of PROTAC degrader candidates consisting of the CK1δ-specific inhibitor scaffold, alkyl and PEG linker motifs with various lengths, and Cereblon (CRBN)-engaging pomalidomide and thalidomide derivatives as E3 ligase binders. We demonstrate that several PROTACs degrade CK1δ/ε in various cells. The most potent PROTAC P1d inhibits the phosphorylation of downstream substrates through CK1δ/ε degradation. We establish the requirement of CUL4A^CRBN and the proteasome for the P1d-mediated degradation of CK1δ/ε. Full article

Doxorubicin (Dox) is a widely used anti-cancer drug. It has proven efficacy against various cancers, although the clinical application of Dox has been limited due to dose-dependent, irreversible, and fatal Dox-induced cardiotoxicity (DIC). The mechanism of DIC remains unclear. p53 plays a key role in DIC via cardiomyocyte loss due to cell death and oxidative stress. Its expression is strictly controlled by post-translational modifications, and its suppression in cardiomyocytes reportedly ameliorates DIC. The ubiquitin system regulates biological processes that are fundamental to the development of cardiovascular diseases. The dysregulation of several ubiquitin E3 ligases is reportedly associated with DIC development through the upregulation of p53. Ubiquitin E3 ligases are classified into four groups; all classes of E3 ligases are involved in p53 degradation. In this review, we focus on recently emerging topics regarding the role of E3 ligases in the regulation of p53 degradation. We also provide an overview of the functional roles of E3 ligases in DIC. Recent reports have identified cardioprotective agents for DIC through ubiquitin E3 ligase-mediated p53 suppression. Here, we present some findings regarding the current development of cardioprotective agents for DIC. These agents may serve as a novel therapeutic target for the treatment of DIC. Full article

Nearly all eukaryotic proteins are turned over by the ubiquitin (Ub)-26S proteasome system (UPS). Despite its broad cellular roles, only a handful of UPS members, particularly the Ub E3 ligases that specifically recognize a protein for ubiquitylation, have been characterized in plants to date. The challenge arises from the transient recognition and rapid degradation of ubiquitylation substrates by the UPS. To tackle this challenge, the emerging biotinylation-based proximity labeling (PL) offers an exciting tool for enriching transient interactors of Ub E3 ligases. In this study, we examined the efficacy of TurboID in identifying substrates of Arabidopsis Skp1-cullin1-F-box (SCF) ligases. We demonstrate that the Arabidopsis Skp1 Like (ASK)1-TurboID is not fully functioning in planta, which led us to discover a novel antagonism between biotinylation and ubiquitylation in regulating protein stability in vivo. This discovery lowers the effectiveness of PL in ubiquitylome studies. However, using one long-known SCF substrate, phytochrome A, we succeeded to apply its TurboID fusion for complementing the far-red-light response of the phyA-211 null mutant allele, suggesting an efficacy of PL in characterizing single ubiquitylation pathways. This study highlighted a limitation of PL in ubiquitylome studies, discovered a new antagonistic pathway of biotinylation, and developed a theoretical guidance for future PL-based characterization of ubiquitylation pathways. Full article

GIGYF2 (Grb10-interacting GYF protein 2) functions as a versatile adaptor protein that regulates gene expression at various levels. At the transcriptional level, GIGYF2 facilitates VCP/p97-mediated extraction of ubiquitylated Rpb1 from stalled RNA polymerase II complexes during DNA damage response. In mRNA surveillance, GIGYF2 participates in ribosome collision-induced quality control, nonsense-mediated decay, no-go decay, and non-stop decay pathways. Furthermore, GIGYF2 interacts with key factors including 4EHP, TTP, CCR4-NOT, DDX6, ZNF598, and TNRC6A to mediate translational repression and mRNA degradation. Additionally, dysregulation of GIGYF2 has been implicated in various pathological conditions, including metabolic diseases, vascular aging, viral infections, and neurodegenerative disorders. This review summarizes the structural and functional characteristics of GIGYF2, highlighting its importance in transcriptional regulation, mRNA surveillance, translational inhibition, and mRNA degradation, while also elucidating its potential as a therapeutic target for disease treatment. Full article

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