Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA.

Lysosomal storage diseases (LSDs) are a group of monogenic diseases characterized by mutations in genes coding for proteins associated with the lysosomal function. Despite the monogenic nature, LSDs patients exhibit variable and heterogeneous clinical manifestations, prompting investigations into epigenetic factors underlying this phenotypic diversity. In this study, we focused on the potential role of epigenetic mechanisms in the pathogenesis of mucopolysaccharidosis IIIB (MPS IIIB) and mucopolysaccharidosis IVA (MPS IVA). We analyzed DNA methylation (5mC) and histone modifications (H3K14 acetylation and H3K9 trimethylation) in MPS IIIB and MPS IVA patients' fibroblasts and healthy controls. The findings revealed that global DNA hypomethylation is present in cell lines for both diseases. At the same time, histone acetylation was increased in MPS IIIB and MPS IVA cells in a donor-dependent way, further indicating a shift towards relaxed open chromatin in these MPS. Finally, the constitutive heterochromatin marker, histone H3K9 trimethylation, only showed reduced clustering in MPS IIIB cells, suggesting limited alterations in heterochromatin organization. These findings collectively emphasize the significance of epigenetic mechanisms in modulating the phenotypic variations observed in LSDs. While global DNA hypomethylation could contribute to the MPS pathogenesis, the study also highlights individual-specific epigenetic responses that might contribute to phenotypic heterogeneity. Further research into the specific genes and pathways affected by these epigenetic changes could provide insights into potential therapeutic interventions for these MPS and other LSDs.

Activity of the Giardia intestinalis proteasome during encystation and its connection with the expression of the cyst wall protein 1 (CWP1).

Giardia is a parasite whose life cycle is composed of two stages: replicative trophozoites, responsible for the symptoms of the disease, and infective cysts, resistant to adverse environments outside of hosts. Proteasomes are multicatalytic peptidase complexes responsible for the specific degradation of proteins in eukaryotic cells. This study assessed the proteasome activity in the trophozoite and during encystation. Strong activation of the proteasome was observed during the differentiation of trophozoites into cysts, reaching its maximum level 24 h after the stimulus. We also found that the Giardia proteasome presents unusual characteristics related to higher eukaryotic proteasomes, making it an eventual therapeutic target. Here we tested the effects on the synthesis of a cyst wall protein by chemical inactivation of the proteasome and by overexpression or partial inhibition of the deubiquitinating protein RPN11 in transfected cells. Moreover, an analysis of the intracellular localization of RPN11 (an integral part of the proteasome regulatory particle) revealed major changes associated with the differentiation of trophozoites into cysts. This evidence further supports the important role of the proteasome in Giardia encystation.

High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.