Littérature scientifique sur le sujet « Ataxin-3, tubulin »

Ataxia and Male Sterility (AMS) is a mutant mouse strain that contains a missense mutation in the coding region of Nna1, a gene that encodes a deglutamylase. AMS mice exhibit early cerebellar Purkinje cell degeneration and an ataxic phenotype in an autosomal recessive manner. To understand the underlying mechanism, we generated neuronal stem cell (NSC) lines from wild-type (NMW7), Nna1 mutation heterozygous (NME), and Nna1 mutation homozygous (NMO1) mouse brains. The NNA1 levels were decreased, and the glutamylated tubulin levels were increased in NMO1 cultures as well as in the cerebellum of AMS mice at both 15 and 30 days of age. However, total β-tubulin protein levels were not altered in the AMS cerebellum. In NMO1 neurosphere cultures, β-tubulin protein levels were increased without changes at the transcriptional level. NMO1 grew faster than other NSC lines, and some of the neurospheres were attached to the plate after 3 days. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1 neurospheres and that the neuronal differentiation potentials were reduced in NMO1 cells compared to NME or NMW7 cells. These results demonstrate that the AMS mutation decreased the NNA1 levels and increased glutamylation in the cerebellum of AMS mice. The observed changes in glutamylation might alter NSC properties and the neuron maturation process, leading to Purkinje cell death in AMS mice.

Increased DNA damage response (DDR) signaling in kidney cyst-lining epithelial cells (CECs) may provide an opportunity for cell-specific therapeutic targeting in autosomal dominant polycystic kidney disease (ADPKD). We hypothesized that inhibiting ataxia telangiectasia mutated (ATM; a proximal DDR kinase) together with low-dose cisplatin overwhelms the DDR response and leads to selective apoptosis of cyst-lining epithelial cells (CECs). Pkd1RC/RC/Atm+/− mice were treated with either vehicle or a single low-dose cisplatin, and the acute effects on CECs (DNA damage and apoptosis) after 72 h and chronic effects on progression (cyst size, inflammation, fibrosis) after 3 weeks were investigated. At 72 h, cisplatin caused a dose-dependent increase in γH2AX-positive nuclei in both CECs and non-cystic tubules but did not cause selective apoptosis in Pkd1RC/RC/Atm+/− mice. Moreover, the increase in γH2AX-positive nuclei was 1.7-fold lower in CECs compared to non-cystic epithelial cells (p < 0.05). Low-dose cisplatin also did not alter long-term disease progression in Pkd1RC/RC/Atm+/− mice. In vitro, human ADPKD cyst-derived cell lines were also resistant to cisplatin (WT9-12: 61.7 ± 4.6%; WT9-7: 64.8 ± 2.7% cell viability) compared to HK-2 (25.1 ± 4.2%), and 3D cyst growth in MDCK cells was not altered. Finally, combined low-dose cisplatin with AZD0156 (an ATM inhibitor) non-selectively reduced γH2AX in both cystic and non-cystic tubular cells and exacerbated cystic kidney disease. In conclusion, these data suggest that CECs are resistant to DNA damage, and that the combination of cisplatin with ATM inhibitors is not an effective strategy for selectively eliminating kidney cysts in ADPKD.

Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd2+) causes renal cancer and is known to inactivate p53. G2/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd2+-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd2+ (50–100 μM) induced DNA damage within 1–6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1–3 h, measured with dihydrorhodamine 123, and G2/M cell cycle arrest at 6 h, which were abolished by the antioxidant α-tocopherol (100 μM). Cd2+-induced G2/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 μM), which relieves G2/M transition block, abolished Cd2+-induced G2 arrest and increased apoptosis. This was accompanied by prevention of Cd2+-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells Cd2+-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G2/M arrest. This may promote survival of premalignant PT cells and Cd2+ carcinogenesis.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid–Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was β-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.

Purpose: A phase I trial was performed with combretastatin A4 phosphate (CA4P), a novel tubulin-binding agent that has been shown to rapidly reduce blood flow in animal tumors. Patients and Methods: The drug was delivered by a 10-minute weekly infusion for 3 weeks followed by a week gap, with intrapatient dose escalation. Dose escalation was accomplished by doubling until grade 2 toxicity was seen. The starting dose was 5 mg/m2. Results: Thirty-four patients received 167 infusions. CA4P was rapidly converted to the active combretastatin A4 (CA4), which was further metabolized to the glucuronide. CA4 area under the curve (AUC) increased from 0.169 at 5 mg/m2 to 3.29 μmol • h/L at 114 mg/m2. The mean CA4 AUC in eight patients at 68 mg/m2 was 2.33 μmol • h/L compared with 5.8 μmol • h/L at 25 mg/kg (the lowest effective dose) in the mouse. The only toxicity that possibly was related to the drug dose up to 40 mg/m2 was tumor pain. Dose-limiting toxicity was reversible ataxia at 114 mg/m2, vasovagal syncope and motor neuropathy at 88 mg/m2, and fatal ischemia in previously irradiated bowel at 52 mg/m2. Other drug-related grade 2 or higher toxicities seen in more than one patient were pain, lymphopenia, fatigue, anemia, diarrhea, hypertension, hypotension, vomiting, visual disturbance, and dyspnea. One patient at 68 mg/m2 had improvement in liver metastases of adrenocortical carcinoma. Conclusion: CA4P was well tolerated in 14 of 16 patients at 52 or 68 mg/m2; these are doses at which tumor blood flow reduction has been recorded.

Potassium bromate (V), (KBrO3) exists as white crystals, crystalline powder or granules. It is highly soluble in water, tasteless and odourless. Potassium bromate is a strong oxidizing agent. In the past it has been used as food additive in flour milling, as an ingredient in fish-paste in Japan, in cheese making, in beer malting, as a component of cold hair wave liquid and an oxidizing compound. Moreover, bromate is formed as a by-product of water disinfection by ozonation and is frequently detected in tap and bottled water. In fact bromate is one of the most prevalent disinfection by-product of surface water. Occupational exposure to potassium bromate occurs mainly in production plants during packaging processes. In Poland, about 1 160 persons were exposed to this compound in 2016. Bromate caused many acute poisonings by accidental ingestion, mainly among children, and more often ingested for tentative suicide by young women, especially hairdressers. In the acute phase of poisoning, gastrointestinal disturbances, irreversible hearing loss, and acute renal failure were observed. Acute renal failure was associated with hemolytic uremic syndrome. There are no data on chronic intoxication of humans by potassium bromate and epidemiological studies on this subject. On the basis of the value of median lethal dose (LD50) per os in rat, potassium bromate has been classified as a compound belonging to the category „Toxic”. Major toxic signs and symptoms in animals after a single intragastric administration of potassium bromate were tachypnea, hypothermia, diarrhea, lacrimation, suppression of locomotor movement, ataxic gait, and animals lying in a prone position. At autopsy the major findings were strong hyperemia of glandular stomach mucosa and congestion of lungs. Microscopically, necrosis and degenerative changes of the proximal tubular epithelium and hearing cells of internal ear were found. It was stated that the compound is not irritating, corrosive or sensitizing. In subchronic and chronic exposure of rodents, potassium bromate led to liver and kidney dysfunction and tubular epithelial damage. Potassium bromate had mutagenic and clastogenic effects. It induced point mutations, structural chromosome aberrations, micronuclei in polychromatic erythrocytes in male mice, DNA oxidative damage by modification of deoxyguanosine to 8-hydroxydeoxyguanosine, and DNA double-strand breakage. Potassium bromate induced neoplasms in rodents and exerted promotion effect in comparison with well-known carcinogens. Besides from preneoplastic changes, expressed by high incidences of renal cell tumors and dysplastic foci, bromate induced solid neoplasms, such as adenomas and adenocarcinomas in a rat kidney and thyroid, and mesotheliomas of peritoneum and tunica vaginalis testis. The European Union classified potassium bromate as a substance that can cause cancer (Group 1.B), whereas IARC classified it as a presumably carcinogenic agent for human (Group 2.B). In principle, effects of bromate on reproduction and ontogenetic development of offspring were not observed. Animal studies suggest that a kidney is a critical organ in the exposure to potassium bromate. The results of subchronic exposure of male rats to potassium bromate administered with drinking water were used to calculate the value of MAC-NDS. The critical effects in kidney were: an increase of organ weight and dose-dependent histopathological alterations defined as epithelium urinary tract hypertrophy. The NOAEL value is 1.5 mg/kg b.w./day. For the calculation of the maximum allowable concentration (MAC) value, 5 uncertainty factors with total value of 24 were used. Based on this estimation it is proposed to accept the MAC-TWA value for potassium bromate at 0.44 mg/m3. The risks of kidney and thyroid cancer in condition of occupational exposure are 2.2 · 10-3 and 0.6 · 10-3, respectively. There is no reason to determine the value of short-term exposure limit (STEL) and the biological exposure index (BEI). „Carc.1.B” notation (carcinogenic substance) was proposed

Abstract Abstract SCI-27 Iron metabolism is regulated in mammals to assure that adequate iron is delivered to the hematopoietic system to support erythropoiesis. In systemic iron metabolism, regulation of both iron uptake from the diet and release from erythrophagocytosing macrophages is coordinated by action of the peptide hormone, hepcidin, which inhibits activity of the iron exporter, ferroportin. In general, high expression of hepcidin diminishes duodenal iron uptake and reduces macrophage iron release, a combination observed in the anemia of chronic disease. Low expression of hepcidin, which is synthesized by hepatocytes and influenced by transferrin receptor 2, HFE, hemojuvelin and bone morphogenetic receptors, facilitates iron uptake. Mutations affecting genes in the hepcidin pathway cause hemochromatosis, characterized by systemic iron overload that affects mainly hepatocytes and cardiac myocytes, but spares the CNS. In contrast, there are several degenerative diseases of the CNS in which neuronal iron overload is prominent and may play a causal role. The underlying pathophysiologies of neuronal brain iron accumulation syndromes remain unclear, even though several causal genes have been identified, including pantothenate kinase 2 and aceruloplasminemia. In some cases, increased iron may be inaccessible, and cells may suffer from functional iron insufficiency, as we propose for animals that lack iron regulatory protein 2. It is also possible that errors in subcellular iron metabolism can lead to mitochondrial iron overload and concomitant cytosolic iron deficiency, a combination observed in Friedreich ataxia, ISCU myopathy, and the sideroblastic anemia caused by glutaredoxin 5 deficiency. In each of these diseases, mitochondrial iron-sulfur cluster assembly is impaired, and it appears that normal regulation of mitochondrial iron homeostasis depends on intact iron-sulfur cluster assembly. Finally, in heme oxygenase 1 deficient animals, macrophages in the spleen and liver die upon erythrophagocytosis, and failure to normally metabolize heme leads to shift of heme iron to proximal tubules and macrophages of the kidney. Thus, treatment of “iron overload” must depend on the underlying causes, and removal of iron is appropriate in hemochromatosis, but more specific forms of therapy are needed for other forms of iron overload. 1. Ye, H. & Rouault, T. A. (2010). Human iron-sulfur cluster assembly, cellular iron homeostasis, and disease. Biochemistry 49, 4945–4956. 2. Zhang, A. S. & Enns, C. A. (2009). Molecular mechanisms of normal iron homeostasis. Hematology Am Soc Hematol Educ Program 207–214. 3. Ye, H., Jeong, S. Y., Ghosh, M. C., Kovtunovych, G., Silvestri, L., Ortillo, D., Uchida, N., Tisdale, J., Camaschella, C. & Rouault, T. A. (2010). Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depleting cytosolic iron in human erythroblasts. J Clin Invest 120, 1749–1761. 4. Ghosh, M. C., Tong, W. H., Zhang, D., Ollivierre-Wilson, H., Singh, A., Krishna, M. C., Mitchell, J. B. & Rouault, T. A. (2008). Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice. Proc Natl Acad Sci U S A 105, 12028–12033. 5. Crooks, D. R., Ghosh, M. C., Haller, R. G., Tong, W. H. & Rouault, T. A. (2010). Posttranslational stability of the heme biosynthetic enzyme ferrochelatase is dependent on iron availability and intact iron-sulfur cluster assembly machinery. Blood 115, 860–869. Disclosures: No relevant conflicts of interest to declare.

Serum- and glucocorticoid-induced kinase 3 (SGK3), which is ubiquitously expressed in mammals, is regulated by estrogens and androgens. SGK3 is activated by insulin and growth factors through signaling pathways involving phosphatidylinositol-3-kinase (PI3K), 3-phosphoinositide-dependent kinase-1 (PDK-1), and mammalian target of rapamycin complex 2 (mTORC2). Activated SGK3 can activate ion channels (TRPV5/6, SOC, Kv1.3, Kv1.5, Kv7.1, BKCa, Kir2.1, Kir2.2, ENaC, Nav1.5, ClC-2, and ClC Ka), carriers and receptors (Npt2a, Npt2b, NHE3, GluR1, GluR6, SN1, EAAT1, EAAT2, EAAT4, EAAT5, SGLT1, SLC1A5, SLC6A19, SLC6A8, and NaDC1), and Na+/K+-ATPase, promoting the transportation of calcium, phosphorus, sodium, glucose, and neutral amino acids in the kidney and intestine, the absorption of potassium and neutral amino acids in the renal tubules, the transportation of glutamate and glutamine in the nervous system, and the transportation of creatine. SGK3-sensitive transporters contribute to a variety of physiological and pathophysiological processes, such as maintaining calcium and phosphorus homeostasis, hydro-salinity balance and acid-base balance, cell proliferation, muscle action potential, cardiac and neural electrophysiological disturbances, bone density, intestinal nutrition absorption, immune function, and multiple substance metabolism. These processes are related to kidney stones, hypophosphorous rickets, multiple syndromes, arrhythmia, hypertension, heart failure, epilepsy, Alzheimer’s disease, amyotrophic lateral sclerosis, glaucoma, ataxia idiopathic deafness, and other diseases.

AbstractMixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102’s cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.

Ataxin-3 (AT3) is a protein consisting of an N-terminal globular Josephin domain and an unstructured C-terminal region that contains a stretch of consecutive glutamines. When its length is beyond a critical threshold, it triggers an inherited neurodegenerative disease, spinocerebellar ataxia type 3. The pathology results from protein misfolding and intracellular accumulation of fibrillar amyloid-like aggregates. Plenty of work has been carried out to elucidate the protein’s physiological role(s), which has shown that AT3 is multifunctional protein. It acts as transcriptional repressor and also carries out its normal function on protein surveillance pathways. In fact, AT3 interacts with RAD23, which shuttles ubiquitinated protein to proteasome, and also with VCP that seems to be involved in the degradation pathway ubiquitin-proteasome dependent and in the retrotranslocation of ERAD substrates. In addition, a recent report suggests that it participates in sorting misfolded protein to aggresomes. Furthermore, it was shown that AT3 binds ubiquitin chains through its ubiquitin-binding motifs and also has, in the Josephin domain, the catalytic triad of thiol proteases that in this protein sustains ubiquitin cleavage. Since a thorough understanding of the protein’s physiological role(s) requires the identification of all the molecular partners interacting with AT3, we pursued this goal by taking advantage of two-dimensional chromatography coupled to tandem mass spectrometry. We found that different AT3 constructs, including the sole Josephin domain, bound α- and β- tubulin from soluble rat brain extracts. Coimmunoprecipitation experiments confirmed this interaction. Also, normal AT3 overexpressed in COS-7 cultured cells partially colocalized with microtubules, whereas an expanded variant only occasionally did so, probably due to aggregation. Furthermore, by surface plasmon resonance (SPR) we determined a dissociation constant of 50–70 nM between AT3 and tubulin dimer, which strongly supports the hypothesis of a direct interaction of this protein with microtubules in vivo. Since misfolded protein is sorted to aggresome via microtubules under conditions where proteasome is overloaded or its function compromised, our finding suggests an involvement of AT3 in directing aggregated protein to aggresome. In further experiments, we assessed by SPR the binding affinity of AT3 with either Lys48-linked or Lys63-linked polyubiquitin chains. We observed that either chains were bound with high affinity by AT3. However, we also found that binding of Lys48-polyubiquitin and tubulin were mutually exclusive, whereas Lys63-polyubiquitin and tubulin could be bound simultaneously. Protein Lys48- and Lys63-polyubiquitination are signals that direct to proteasome and aggresome, respectively. Thus, our findings point to a model whereby AT3 sorts tagged protein to either goals, depending on the type of ubiquitin bound. In the former case, AT3 cannot bind to tubulin so it is prevented from being directed to aggresome; in the latter it sorts misfolded protein to aggresome. In keeping with this hypothesis, we also found that AT3 binds to histone deacetylase 6 (HDAC6) with nanomolar affinity. Actually, the latter protein is indispensable for aggresome formation, as it also binds Lys63-polyubiquitylated proteins and interacts with the dynein complex, which transports misfolded protein cargo to aggresomes via microtubules. Finally, we also investigated the mode of interaction between AT3 and tubulin by producing several truncated AT3 variants, and determining their affinity towards tubulin. These studies highlight the occurrence of three different tubulin-binding sites: in the Josephin domain, in the disordered stretch upstream of the polyQ tract, and at the C-terminus, at the very end of the polypeptide chain.

Ataxin-3 (AT3) is a deubiquitinating enzyme that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. It consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. Regarding its physiological role, it has ubiquitin hydrolase activity implicated in the function of the ubiquitin-proteasome system, but also plays a role in the pathway that sorts aggregated protein to aggresomes via microtubules. In the first part of this work, we further investigated its function(s) by taking advantage of Small Angle X-ray Scattering (SAXS) and Surface Plasmon Resonance (SPR). We demonstrated that an AT3 oligomer consisting of 6-7 subunits tightly binds to the tubulin hexameric oligomer at the level of three distinct tubulin-binding regions, one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, one of the components of the transport machinery that sorts protein to the aggresome. In the second part of this work, we have investigated the mechanisms of AT3 cytotoxicity triggered by expanded variants. For this purpose, we used Saccharomyces cerevisiae as a eukaryotic cellular model. We expressed a wild type (Q26), a pathogenic (Q85) and a truncated (291Δ) variant of the protein. The expanded form caused reduction in viability, accumulation of reactive oxygen species, imbalance of the antioxidant defense system and loss in cell membrane integrity. An AT3 variant truncated upstream of the polyQ also exerted a detrimental effect on cell growth and similar cytotoxicity, although to a lesser extent, which points to the involvement of also non-polyQ regions in cytotoxicity. Finally, we sought to evaluate the effects of tetracycline and epigallocatechin-3-gallate (EGCG), two well-known inhibitors of amyloid aggregation, on AT3 fibrillogenesis and cytotoxicity. We observed that tetracycline does not apparently change the aggregation mode, as supported by Fourier Transform Infrared spectroscopy and Atomic Force Microscopy data, but slightly retards further aggregation of the earliest soluble oligomers. In contrast, EGCG apparently increases the aggregation rate but also leads to the formation of off-pathway, non-amyloid, final aggregates. Despite these different effects, co-incubation of the AT3 with either compounds resulted in significantly lower cytotoxicity during AT3 aggregation.