CLUH, an RNA-binding protein (RBP), is known to regulate liver metabolism under conditions of acute nutrient stress. However, its role in metabolic dysfunction-associated steatotic liver disease (MAFLD) and metabolic dysfunction-associated steatohepatitis (MASH) remains unknown. Here, we show that CLUH is downregulated in the methionine-choline-deficient (MCD) mouse model of MASH, as well as in MAFLD and MASH patients. Whereas overexpression of CLUH alleviates hepatic lipid accumulation, fibrosis, and serum AST and ALT levels induced by the MCD diet. Mechanistically, we found that CLUH regulates mitochondrial health, endoplasmic reticulum stress, and cell death by regulating Agxt2 expression. Collectively, our findings identify CLUH as a novel regulator of ER homeostasis and a potential target to treat MASH.

Delivering functional nanobodies intracellularly is a powerful method for studying cellular pathways and tracking protein dynamics in live cells. Recently, the field has advanced towards interrogating pathological targets to modulate cellular function. This review summarises current research employing diverse strategies for cellular uptake of nanobodies and subsequent antigen recognition. We specifically emphasise the potential of cell-permeable nanobodies as functional therapeutics in relevant diseases, such as cancer, respiratory diseases and neurological disorders. Lastly, we discuss the current challenges, translational clinical efforts and future perspectives for cell-permeable nanobodies.

Peroxisomes are cellular organelles that contribute to several crucial metabolic processes. Despite being present in almost all eukaryotic cells, these organelles were regarded as little more than the cell's 'rubbish bin' for years following their discovery. The trigger that stimulated research into the roles of peroxisomes in human pathophysiology was the identification of peroxisomal defects as the cause of a group of inherited metabolic disorders. In this latest article in our series on patient advocacy, we interview Andrew Longenecker, CEO of PBD Project, and Dr. Einat Zalckvar, who has carried out research into yeast and mammalian peroxisomes and peroxisomal disorders for over a decade.

Hedgehog (Hh) signaling is frequently activated in cancers, such as basal cell carcinoma and medulloblastoma. Although Smoothened (SMO) inhibitors such as vismodegib and sonidegib are used to treat Hh-driven tumors, their efficacy is often limited by drug-resistant SMO mutations. Here, we investigated whether targeting DYRK2, a positive regulator of Hh signaling downstream of SMO, suppresses Hh signaling driven by the drug-resistant SMO[W535L] mutation. Genetic depletion and pharmacological inhibition of DYRK2 suppressed Hh target gene expression in SMO[W535L]-expressing cells. In contrast to vismodegib, DYRK2 inhibition reduced oncosphere formation in SMO[W535L]-expressing medulloblastoma cells. These findings identify DYRK2 as a regulatory node downstream of SMO and demonstrate that targeting DYRK2 represents a strategy to overcome resistance to SMO inhibitors in Hh-driven tumors.

The ever-growing pollution, persistence, and recalcitrance of toxic aromatic compounds require highly efficient biocatalytic solutions that outperform natural microbial metabolism. Rieske (2Fe-2S)-type dioxygenases (ROs) are a promising class of enzymes known to degrade various toxic aromatic compounds by hydroxylating aromatic rings, thereby facilitating their cleavage. ROs include biphenyl dioxygenase (BPDO), phthalate dioxygenase (PDO), terephthalate dioxygenase (TPDO), and isophthalate dioxygenase (IPDO), which degrade their respective substrates into the corresponding cis-dihydrodiols. Although they have narrow substrate-specificity constraints, they can be customized by redesigning the active-site architecture and substrate-access tunnels to generate variants with a broader substrate range and enhanced catalytic efficiency. Rational engineering of IPDO has demonstrated the advantage of introducing targeted mutations to expand substrate scope to include multiple phthalate regioisomers without compromising catalytic performance. Similarly, mutational studies of BPDO have shown that substituting interacting residues can increase conformational flexibility and accommodate structurally diverse substrates, such as polychlorinated biphenyls. This review analyzes the rational engineering of various Rieske dioxygenase enzymes, based on structural studies, to expand their catalytic range for the biodegradation of toxic aromatic pollutants.

Eukaryotic vacuolar H[+]-ATPase (V-ATPase) is a multisubunit rotary proton pump that acidifies intracellular compartments, energized by ATP hydrolysis. Despite extensive structural data, our understanding of the molecular basis of its conformational changes and functionally critical sites remains incomplete. Using elastic network models, we performed spectral decomposition of the intrinsic modes of motion of human V-ATPase, identifying hinge residues governing its most cooperative structural rearrangements. Most of these hinges are evolutionarily conserved or co-evolutionarily coupled, underscoring their mechanical role in the allosteric machinery. Experimentally observed structural transitions, including rotary state changes, V1V0 dissociation, and autoinhibition, are encoded in the softest intrinsically accessible modes. These structure-encoded soft modes provide conformational paths to alternate functional states and highlight new sites for pharmacological interventions.

AADC deficiency is a rare autosomal recessive disorder characterized by motor/neurodevelopmental symptoms. Approximately one-third of patients experience recurrent hypoglycemia, with or without neurological involvement. To investigate whether specific variants identified in hypoglycemic patients underlie this metabolic phenotype, we characterized three amino-terminal variants. The crystal structure of p.Asp15Gly revealed disruption of an inter-subunit electrostatic network stabilizing the enzyme interface. Computational analyses predicted that p.Val33Leu and p.Gly36Arg perturb an essential catalytic element. Functional studies showed that p.Asp15Gly retained substantial catalytic activity and coenzyme affinity despite reduced stability, whereas p.Val33Leu and p.Gly36Arg exhibited increasing structural destabilization, reduced coenzyme affinity, and severe catalytic defects. These findings reveal distinct functional roles of the amino-terminal-domain, providing a molecular basis for the clinical variability of AADC deficiency.

The increasing prevalence of viral diseases represents a major threat to global human health, highlighting the urgent need for advanced tools to study viral infections, improve diagnostics, and develop effective antiviral therapies. Antibodies are central to the immune response against viruses and are widely used in research and diagnostics; however, their structural complexity and manufacturing process limit their areas of applications. Single-domain antibodies-better known as nanobodies-derived from heavy chain-only antibodies found in camelids have emerged as a promising alternative. Their unique characteristics-including small size, high stability, the capacity to access cryptic epitopes, and functionality within intracellular environments-make them particularly valuable for viral research. This review highlights recent developments of nanobodies for viral research, diagnostics, and therapeutic applications; discusses their key advantages and limitations; and briefly outlines the emerging role of artificial intelligence-based technologies in facilitating their development to address the expanding burden of viral diseases.

Renal fibrosis is a hallmark of chronic kidney disease (CKD), but effective antifibrotic therapies are lacking. Pentraxin 3 (PTX3) is markedly upregulated in CKD patients and in a unilateral ureteral obstruction (UUO) model, correlating with renal injury, extracellular matrix accumulation, partial epithelial-mesenchymal transition markers, and TGF-β1 signaling. Genetic deletion of Ptx3 significantly attenuates UUO-induced fibrosis. Mechanistically, PTX3 promotes transcription of LTBP-1 and fibronectin, facilitating latent TGF-β1 activation and inducing a profibrotic partial EMT program in tubular epithelial cells without lineage conversion. Importantly, blockade of the PTX3/CD44 axis using a neutralizing αPTX3 antibody reduced serum creatinine levels and renal fibrosis, identifying PTX3 as a promising therapeutic target for CKD.

LigE is a periplasm-targeted ATP-dependent DNA ligase found in many Gram-negative bacteria, including Neisseria gonorrhoeae. Although LigE has been shown to have a role in biofilm formation, many LigE-possessing bacteria are also naturally competent, suggesting a possible role in transformation with extracellular DNA. Here, we demonstrate that LigE participates in competence by increasing transformation with nuclease-damaged extracellular DNA that contains single-stranded or cohesive breaks, and that this activity is dependent on its secretion signal peptide, indicating an extra-cytoplasmic location of activity. We show that increased transformation with this restricted DNA is ATP-dependent; however, the physiological source of ATP in the natural host remains unknown. Our findings highlight a potential role of Ngo-LigE in Neisseria gonorrhoeae competence and horizontal gene transfer.

Progeria is an extremely rare genetic disease in which affected individuals age rapidly due to a specific mutation in the LMNA gene, which results in the production of progerin, an abnormal protein that damages cell nuclei and causes progressive multi-organ disease. AIProSaB is an Italian association founded by Sammy Basso, who was diagnosed with progeria at the age of two, and by his parents. The association supports research, promotes experimental therapies, and provides information for families and clinicians, with a strong emphasis on understanding LMNA/progerin biology and developing targeted treatments and gene-editing approaches. As part of our series on patient advocacy, FEBS Letters interviews Dr. Giovanna Lattanzi, a researcher at the CNR Institute of Molecular Genetics 'Luigi Luca Cavalli-Sforza' in Bologna on the aims, achievements, and activities of AIProSaB.

Lysophagy selectively eliminates damaged lysosomes to preserve lysosomal homeostasis, but its regulatory mechanisms remain incompletely understood. Here, we identify dipeptidyl peptidase 7 (DPP7), a lysosomal serine protease, as a regulator of lysophagy. Inhibition or depletion of DPP7 impaired clearance of LLOMe-damaged lysosomes in HepG2 cells, as evidenced by sustained galectin-3 puncta and reduced lysophagic flux. DPP7 inhibition did not block TFEB activation; however, it markedly reduced ubiquitin accumulation on damaged lysosomes and attenuated the recruitment of SQSTM1 and LC3. This defect was accompanied by impaired lysosomal re-acidification and reduced maturation of cathepsin D and L. Together, our findings identify DPP7 as a novel modulator of ubiquitin-dependent lysophagy and reveal an unexpected role for a lysosomal peptidase in lysosomal quality control.

Cattle produce a subset of antibodies (Abs) with unique architecture. These immunoglobulins feature an ultralong CDR-H3 (uCDR-H3) composed of up to about 70 amino acids that protrudes from the scaffold and folds into a distinct stalk-and-knob structure. The knob is a disulfide-rich mini-domain that contains all paratope residues for antigen recognition. Due to their vast structural diversity and low molecular weight, cattle-derived knob paratopes have emerged as promising building blocks for engineering next-generation Abs. In this review, we discuss the structural features of uCDR-H3 Abs alongside engineering strategies of cattle-derived knob architectures, including stand-alone knob peptides and knob engraftments to generate bispecific antibodies (bsAbs).

BRCA2 is a central tumor suppressor protein best known for its roles in homologous recombination and replication fork stability. Increasing evidence, however, positions BRCA2 at the interface of transcription and replication, where it preserves genome stability under transcription-associated stress. BRCA2 regulates R-loop homeostasis, transcription-replication conflict resolution, and RNA polymerase II dynamics. Its loss leads to R-loop accumulation, transcriptional pausing, and elevated transcription-replication head-on collisions, particularly in highly transcribed regions. BRCA2 also promotes the processing of RNA-associated DNA lesions, preventing toxic intermediates during S phase. Through interactions with factors such as PAF1, PCAF, and DDX5, BRCA2 coordinates transcription, chromatin regulation, and DNA-RNA hybrid resolution, linking these processes to inflammatory and stress-responsive transcriptional programs, including NF-κB signaling. In this review, we focus on the emerging roles of BRCA2 in transcription-related pathways, including R-loop homeostasis, resolution of transcription-replication conflicts, RNA polymerase II dynamics, and broader transcriptional regulation.

Skin fibrosis is marked by extracellular matrix accumulation and dermal white adipose tissue (DWAT), resulting in tissue stiffening. We show that sustained Wnt activation in mature dermal adipocytes induces lipodystrophy and promotes fibrotic remodeling via oxidative stress. Meta-analysis of early systemic sclerosis skin transcriptomes reveals increased reactive oxygen species (ROS) production and lipid peroxidation. In cultured mouse dermal adipocytes, Wnt activation triggers a robust increase in ROS and lipid peroxidation, which is suppressed by scavengers. In vivo, Wnt activation elevates oxidative stress in DWAT, and N-acetyl-L-cysteine scavenger protects from DWAT lipodystrophy and dermal collagen accumulation. These findings identify a Wnt-ROS-lipid peroxidation axis as a key driver of lipodystrophy and skin fibrosis, highlighting a potential therapeutic target.