The FEBS journal最新文献

Congenital dyserythropoietic anemia type I (CDA-I) is a rare hereditary disease marked by ineffective erythropoiesis, a characteristic spongy heterochromatin structure in erythroblasts, and mutations in the genes CDAN1 and CDIN1, which encode the proteins Codanin1 and CDIN1. Codanin1 regulates histone shuttling via the chaperone ASF1, yet the role of CDIN1 in CDA-I pathology remains unclear. Notably, CDIN1 is known to interact directly with the C-terminus of Codanin1. Although mutations in both genes are critical to the disease phenotype, their molecular-level effects have not been fully elucidated. Here, we present a comprehensive structural and functional analysis of the CDIN1-Codanin1 C-terminus complex. Using complementary biophysical techniques, we show that CDIN1 and Codanin1 C-terminus form a high-affinity heterodimeric complex with equimolar stoichiometry. We further delineate the essential interacting regions of CDIN1 and Codanin1. We demonstrate that CDA-I-associated mutations in either protein disrupt the CDIN1-Codanin1 interaction, suggesting a potential molecular mechanism underlying the disease.

Proteostasis maintains the balance between protein synthesis, folding, and degradation within the endoplasmic reticulum (ER). This quality-control system ensures that proteins undergo proper post-translational modifications-such as PDI-ERO1-mediated oxidative folding and STT3-dependent N-glycosylation-so that only correctly folded proteins proceed through the secretory pathway. Impairment of protein load, folding capacity, or degradation via the ER-associated degradation (ERAD) pathway leads to the accumulation of unfolded proteins, triggering ER stress and activating the unfolded protein response (UPR), which, in the first instance, is an adaptive signaling network designed to restore homeostasis by adjusting protein synthesis, enhancing folding capacity, and promoting the clearance of misfolded proteins. During ER stress, the ER undergoes morphological and functional remodeling to manage the increased folding burden, including an increase of ER-mitochondria contact sites (ERMCs). These nanometric junctions (~10-100 nm) facilitate lipid and metabolite exchange and mediate calcium and reactive oxygen species signaling to support cellular metabolism. However, chronic ER stress can further tighten ERMCs, leading to calcium overload, mitochondrial dysfunction, and apoptosis. This review examines the core mechanisms underlying ER proteostasis in the context of ER stress and explores how ER stress first boosts mitochondrial activity and later impairs it through ERMCs, contributing to cell death and disease. Finally, emerging therapeutic strategies aimed at restoring proteostasis and modulating the dynamics of ERMCs are highlighted as promising interventions for conditions, such as cancer and congenital myopathies, where ER and mitochondrial dysfunction play central roles in pathogenesis.

The Nedd4 subfamily of HECT E3 ligases is a ubiquitous group of 10 enzymes that share the same domain structure, consisting of a C2 domain, several WW domains and a catalytic HECT domain. Over the past decade, significant progress has been made in characterizing the molecular details of their activity and regulation. Studies have shown that, in the inactive state, the HECT domain is shielded by its N-terminal domains, thereby blocking access to the active site. The catalytic functions of Nedd4 enzymes include accepting ubiquitin molecules from ubiquitin-conjugating enzymes, transferring them to substrates, and generating diverse polyubiquitin chains. The modulation of Nedd4 enzyme activity involves mechanisms that facilitate enzymatic activation, relay binding to components of the enzymatic cascade, and enable (auto)ubiquitination. This minireview provides a comprehensive overview of the structural features distinguishing the inactive and active conformations in this group of E3 ligases, while underscoring the need for further research necessary to develop pharmaceutical solutions targeting pathological conditions rooted in Nedd4 dysfunction.

Wnt signaling regulates metazoan development and homeostasis, in part by β-catenin-dependent activation and repression of a large number of genes. However, Wnt signaling also regulates genes independent of β-catenin, genes that are less well-characterized. In this study, using a pan-Wnt inhibitor, we performed a comprehensive transcriptome analysis in a Wnt-addicted orthotopic cancer model to delineate the β-catenin-dependent and independent arms of Wnt signaling. We find that while a large percentage of Wnt-regulated genes are regulated by β-catenin, 10% of these genes are regulated independent of β-catenin. Interestingly, a large proportion of these β-catenin-independent genes are Wnt-repressed. Among the β-catenin-dependent genes, more than half are repressed by β-catenin. We used this dataset to investigate the mechanisms by which Wnt/β-catenin signaling represses gene expression, revealing the role of a cis-regulatory motif, the negative regulatory element (NRE). The NRE motif is enriched in the promoters of β-catenin repressed genes and is required for their repression. This provides a comprehensive analysis of the β-catenin-independent arm of the Wnt signaling pathway in a cancer model and suggests that a cis-regulatory grammar may determine Wnt-dependent gene activation versus repression.

Proteases hydrolyze the amide bond of a polypeptide chain, which can influence protein synthesis and function. This activity has been predominantly observed in the post-translational processing of lysyl oxidase (LOX) and lysyl-oxidase-like (LOXL) family proteins, which are indispensable for the remodeling of the extracellular matrix (ECM). Of the four classes, metallo- and serine proteases are known to catalyze the hydrolysis of pro-LOX and pro-LOXL family proteins into their processed forms. These LOX family proteins play instrumental roles in ECM remodeling by oxidatively deaminating the lysine and 5-hydroxy lysine residues, primarily in collagens and elastin, thus facilitating the formation of lysyl-derived covalent crosslinking that stabilizes the extracellular matrix assembly. Previous studies have established the presence of different proteolytic sites and the corresponding proteases for different LOX family proteins. However, the underlying mechanism of this protease-mediated processing of the LOX family in fibrotic tissue remodeling has remained elusive. In this review, we summarize the critical role of ECM crosslinking reactions catalyzed by the LOX family and provide an overview of proteolytic sites and corresponding proteases in fibrosis. Moreover, we discuss the potential catalytic mechanisms of bone morphogenetic protein-1, a metalloprotease, and Factor Xa, a serine protease, on LOX and LOXL2, respectively.

Signal transduction processes often involve membrane-associated proteins allowing facilitated diffusion of reactive partners in a phospholipid bilayer plane. A benchmark example is phototransduction, taking place at the disk membranes of vertebrate rod and cone outer segments. Long-wavelength sensitive cones in night migratory songbirds harbor another sensory signaling pathway. These birds can detect the Earth's magnetic field probably utilizing a radical-pair mechanism based on a photosensitive process in a cryptochrome protein. The isoform cryptochrome 4a in European robin is discussed as a prime magnetoreceptor candidate based on its photochemistry. However, cryptochrome 4a needs to have a fixed position on the membrane to operate as a magnetic field detector. We employed surface plasmon resonance to immobilize phospholipid bilayers on a sensor chip surface to investigate critical protein-lipid interaction processes. One possible interaction partner of ErCry4a is the myristoylated G-protein α-subunit from European robin cone cells. The G protein bound to lipid bilayers with moderate-to-high affinity, consistent with a combination of hydrophobic and electrostatic interactions. ErCry4a could also interact with a pure lipid bilayer, but also with bilayers that have the myristoylated G-protein α-subunit attached. Both binding processes occurred with small differences in affinities, displaying K_D values in a range from 51 nm to 130 nm. Our results point to the importance of the myristoyl group for the interaction process and agree with a model where Gtα molecules could diffuse to ErCry4a, forming a high affinity complex for downstream signaling in magnetoreception.

Rickettsia parkeri rickettsiosis is an emerging tick-borne disease in humans. The bacterium R. parkeri is primarily transmitted to humans by the Gulf Coast tick, Amblyomma maculatum. Upon transmission to the host, R. parkeri infects and multiplies in host endothelial cells. In this study, we provide evidence that R. parkeri modulates the host nicotinamide adenine dinucleotide (NAD+) pathway for its survival in endothelial cells. Analysis performed with arrays containing antibodies against proteins involved in different metabolic pathways revealed increased levels of BST1 (an NAD+-dependent molecule) in R. parkeri-infected endothelial cells compared to the levels noted in uninfected controls. Quantitative PCR analysis further validated antibody array results. We noted modulation of several NAD+ pathway genes and increased reactive oxygen species (ROS) production upon R. parkeri infection in endothelial cells. Treatment of endothelial cells with NAD+ before R. parkeri infection but not postinfection significantly increased ROS levels that affected the bacterial burden. In contrast, we noted increased R. parkeri loads and reduced ROS levels in endothelial cells when bacteria were pre-incubated with NAD+ before infection. Furthermore, siRNA-mediated silencing of bst1 expression increased bacterial loads, reduced ROS levels, and affected the expression of NAD+ metabolic pathway genes. Collectively, these results not only elucidate the importance of the host NAD+ metabolic pathway in limiting R. parkeri infection by ROS production but also suggest its therapeutic role in preventing this and perhaps other rickettsial infections of medical importance.

Notch signalling is an evolutionarily conserved signalling pathway that directs cell growth and differentiation across multiple tissue types, and its regulation must be controlled across the lifespan. Aberrant Notch signalling due to genetic mutations that occur within the negative regulatory region of the Notch 1 gene is linked to the development of acute T-cell leukaemia in humans. This discovery has led to a concerted effort to understand how Notch receptor signalling is regulated in mammalian cells. Liu et al. have developed a range of novel peptide inhibitors that target the heterodimerization domain within the negative regulatory region of the Notch receptor. They show that the peptide inhibitors are specific to a Notch receptor paralogue. The possible biological and therapeutic consequences are discussed.

The metastatic progression of melanoma, a highly immunogenic cancer, is often associated with constitutive expression of major histocompatibility complex (MHC) class II molecules. Their engagement leads to increased expression and activation of signaling proteins, kinases, and adhesion receptors. Melanoma cells release extracellular vesicles (EVs) that are involved in the metastatic progression of melanoma through modifying the tumor microenvironment. Here, we report that MHC class II molecules, when constitutively expressed on melanoma cells, drive the role of EVs in regulating immune cell function, melanoma metastasis, and tumor microenvironment remodeling. In particular, we observed an increased localization of HLA-DRα, CAM receptors, PD-L1, and STAT3 signaling proteins in EVs. Using co-culture experiments, we demonstrate the EV-induced apoptosis of PBMCs, as well as the increased migration of fibroblasts and melanoma cells in response to MHC class II-activated signaling. Overall, these results highlight a complex and dynamic interplay between fibroblasts, tumor cells, and EVs, which, as key mediators of cell communication, establish a paracrine and autocrine signaling circuit crucial for regulating tumor cell and fibroblast migration. Our results demonstrate that MHC class II-activated signaling is critical for melanoma progression, driving enhanced metastatic dissemination and immune evasion via extracellular vesicles in the tumor microenvironment.

ADP-ribosyltransferases PARP1 and PARP2 are involved in DNA repair mechanisms and play a major role in detecting DNA damage. PARP1/2 enzymes transfer the ADP-ribosyl moiety from NAD⁺ to DNA damage response proteins, histones and to itself, activating the DNA repair cascade. Recent studies have shown that histone PARylation factor 1 (HPF1) forms a joint active site with the catalytic domain of PARP1/2 and alters their target modification site from glutamate/aspartate to serine. In this work, we identified key serine residues within the N-terminus of full-length human PARP2 that are the main targets of automodification. We demonstrated this using site-directed mutagenesis, gel-based PARylation assays of automodification reactions, and by measuring the release of PARP2 from the DNA damage site using a fluorescence polarization assay. We show that, in the presence of HPF1, PARP2 serine 8 and serine 73 are predominantly ADP-ribosylated and serine 8, which is present in the two human PARP2 isoforms, is the major site for PARP2 automodification. Our results provide insight into the mechanistic role of the N-terminus of PARP2 in the PARylation-dependent release of PARP2 from DNA damage sites.