Selectivity of Hydroxamate- and Difluoromethyloxadiazole-Based Inhibitors of Histone Deacetylase 6 In Vitro and in Cells.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

Mitochondrial respiration supports autophagy to provide stress resistance during quiescence.

Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.

Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX.

Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.

The avian retroviral receptor Tva mediates the uptake of transcobalamin bound vitamin B12 (cobalamin).

The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl).Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV.IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.

Desminopathy: Novel Desmin Variants, a New Cardiac Phenotype, and Further Evidence for Secondary Mitochondrial Dysfunction.

Background: The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. We aimed to describe the prevalence, phenotypic expression, and mitochondrial function of individuals with putative disease-causing desmin (DES) variants identified in patients with an unexplained etiology of cardiomyopathy. Methods: A total of 327 Czech patients underwent whole exome sequencing and detailed phenotyping in probands harboring DES variants. Results: Rare, conserved, and possibly pathogenic DES variants were identified in six (1.8%) probands. Two DES variants previously classified as variants of uncertain significance (p.(K43E), p.(S57L)), one novel DES variant (p.(A210D)), and two known pathogenic DES variants (p.(R406W), p.(R454W)) were associated with characteristic desmin-immunoreactive aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel DES variant p.(Q364H) had a decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal muscle biopsy and presented with familial left ventricular non-compaction cardiomyopathy (LVNC), a relatively novel phenotype associated with desminopathy. An assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial/skeletal muscle specimens, which was in case of myocardial succinate respiration more profound than in other cardiomyopathies. Conclusions: The presence of desminopathy should also be considered in individuals with LVNC, and in the differential diagnosis of mitochondrial diseases.

Histone Deacetylase 11 Is a Fatty-Acid Deacylase.

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M-1 s-1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 µM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty-acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.