Biochemical Journal最新文献

Despite extensive research, strategies to effectively combat breast cancer stemness and achieve a definitive cure remains elusive. CD44, a well-defined cancer stem cell marker is reported to promote breast cancer tumorigenesis, metastasis, and chemoresistance. However, mechanisms leading to its enhanced expression and function is poorly understood. Here, we demonstrate that USP10 positively regulates CD44 protein levels and its downstream actions. While USP10 depletion prominently downregulates CD44 protein levels and functions, its overexpression significantly enhances CD44 protein levels, leading to enhanced cluster tumor cell formation, stemness, and metastasis in breast cancer cells both in vitro and ex vivo in primary human breast tumor cells. USP10 interacts with CD44 and stabilizes it through deubiquitination both in breast cancer cell lines and human breast cancer-derived primary tumor cells. Stabilized CD44 shows enhanced interaction with cytoskeleton proteins Ezrin/Radixin/Moesin and potently activates PDGFRβ/STAT3 signalling which are involved in promoting CSC traits. Using USP10 stably expressing 4T1 cells, we further demonstrate that the USP10-CD44 axis potently promotes tumorigenicity in vivo in mice, while simultaneous depletion of CD44 in these cells renders them ineffective. In line with these findings, we further showed that inhibition of USP10 either through RNAi or the pharmacological inhibitor Spautin-1 significantly mitigated CD44 levels and its downstream function ex vivo in primary breast tumor cells. Finally, we demonstrated that primary breast tumor cells are more susceptible to chemotherapy when co-treated with USP10 inhibitor indicating that the USP10-CD44 axis could be an attractive therapeutic target in combination with chemotherapy in CD44 expressing breast cancers.

Bacteria evolve mechanisms to compete for limited resources and survive in new niches. Here we study the mechanism of isethionate import from the sulfate-reducing bacterium Oleidesulfovibrio alaskensis. The catabolism of isethionate by Desulfovibrio species has been implicated in human disease, due to hydrogen sulfide production, and has potential for industrial applications. O. alaskensis employs a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM) to import isethionate, which relies on the substrate-binding protein (OaIseP) to scavenge isethionate and deliver it to the membrane transporter component (OaIseQM) for import into the cell. We determined the binding affinity of isethionate to OaIseP by isothermal titration calorimetry (ITC), KD = 0.95 µM (68% CI = 0.6-1.4 µM), which is weaker compared to other TRAP substrate-binding proteins. The X-ray crystal structures of OaIseP in the ligand-free and isethionate-bound forms were obtained and showed that in the presence of isethionate, OaIseP adopts a closed conformation whereby two domains of the protein fold over the substrate. We serendipitously discovered two crystal forms with sulfonate-containing buffers (HEPES and MES) bound in the isethionate-binding site. However, these do not evoke domain closure, presumably because of the larger ligand size. Together, our data elucidate the molecular details of how a TRAP substrate-binding protein binds a sulfonate-containing substrate, rather than a typical carboxylate-containing substrate. These results may inform future antibiotic development to target TRAP transporters and provide insights into protein engineering of TRAP transporter substrate-binding proteins.

The active site Cys residue in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to oxidation by hydrogen peroxide (H2O2), with this resulting in enzyme inactivation. This re-routes the carbon flux from glycolysis to the pentose phosphate pathway favoring the formation of NADPH and synthetic intermediates required for antioxidant defense and repair systems. Consequently, GAPDH inactivation serves as a redox switch for metabolic adaptation under conditions of oxidative stress. However, there is a major knowledge gap as to how GAPDH is efficiently oxidized and inactivated, when the increase in intracellular H2O2 is modest, and there is a high concentration of alternative (non-signaling) thiols and efficient peroxide removing systems. We have therefore explored whether GAPDH inactivation is enhanced by two factors of in vivo relevance: macromolecular crowding, an inherent property of biological environments, and the presence of bicarbonate, an abundant biological buffer. Bicarbonate is already known to modulate H2O2 metabolism via formation of peroxymonocarbonate. GAPDH activity was assessed in experiments with low doses of H2O2 under both dilute and crowded conditions (induced by inert high molecular mass polymers and small molecules), in both the absence and presence of 25 mM sodium bicarbonate. H2O2-induced inactivation of GAPDH was observed to be significantly enhanced under macromolecular crowding conditions, with bicarbonate having an additional effect. These data strongly suggest that these two factors are of major importance in redox switch mechanisms involving GAPDH (and possibly other thiol-dependent systems) within the cellular environment.

Glycation-induced stress (G-iS) is a physiological phenomenon that leads to the formation of advanced glycation end-products (AGEs), triggering detrimental effects such as oxidative stress, inflammation, and damage to intracellular structures, tissues, and organs. This process is particularly relevant because it has been associated with various human pathologies, including cancer, neurodegenerative diseases, and diabetes. As therapeutic alternatives, coordination compounds with antioxidant activity show promising potential due to their versatility in attenuating oxidative stress and inflammation. Herein, we investigated the antioxidant-related protective potential of a series of complexes: [Cu(II)(BMPA)Cl2] (1), [Fe(III)(BMPA)Cl3] (2), and [Cl(BMPA)MnII-(μ-Cl)2-MnII(BMPA)-(μ-Cl)- MnII(BMPA)(Cl)2]•5H2O  (3), all synthesized with the ligand bis-(2-pyridylmethyl)amine (BMPA) in Saccharomyces cerevisiae exposed to G-iS caused by methylglyoxal (MG). Pre- treatment with complexes 1 - 3 proved highly effective, increasing yeast tolerance to G-iS and attenuating mitochondrial dysfunction. This observed phenotype appears to result from a reduction in intracellular oxidation, lipid peroxidation levels, and glycation. Additionally, an increase in the activity of the antioxidant enzymes superoxide dismutase and catalase was observed following treatment with complexes 1 - 3. Notably, although complexes 1 - 3 provided significant protection against oxidative stress induced by H2O2 and menadione, their protective role was more effective against MG-induced glycation stress. Our results indicate that these complexes possess both antiglycation and antioxidant properties, warranting further investigation as potential interventions for mitigating glycation and oxidative stress-related pathologies.

Lamins form a proteinaceous meshwork as a major structural component of the nucleus. Lamins, along with their interactors, act as determinants for chromatin organization throughout the nucleus. The major dominant missense mutations responsible for autosomal dominant forms of muscular dystrophies reside in the Ig fold domain of lamin A. However, how lamin A contributes to the distribution of heterochromatin and balances euchromatin, and how it relocates epigenetic marks to shape chromatin states, remains poorly defined, making it difficult to draw conclusions about the prognosis of lamin A-mediated muscular dystrophies.In the first part of this report, we identified the in-vitro organization of full-length lamin A proteins due to two well-documented Ig LMNA mutations, R453W and W514R. We further demonstrated that both lamin A/C mutant cells predominantly expressed nucleoplasmic aggregates. Labelling specific markers of epigenetics allowed correlation of lamin A mutations with epigenetic mechanisms. In addition to manipulating epigenetic mechanisms, our proteomic studies traced diverse expressions of transcription regulators, RNA synthesis and processing proteins, protein translation components, and posttranslational modifications. These data suggest severe perturbations in targeting other proteins to the nucleus.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.

PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.