12 research outputs found
Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B
No full textPathogenic mutations in the copper transporter ATP7B have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease
Functional analysis of syntrophic LCFA-degrading microbial ecosystems
No full textTese de doutoramento em Engenharia Química e BiológicaAnaerobic degradation of long-chain fatty acids (LCFA), is not yet completely
understood. Previous studies suggest that different microorganisms might be involved in
the degradation of saturated and unsaturated LCFA and that these compounds inhibit
severely the microbial activity, especially the methanogenic activity. In this study, the
toxic effect of saturated- (palmitate (C16:0) and stearate (C18:0)) and unsaturated-LCFA
(oleate (C18:1)), towards pure cultures of hydrogenotrophic methanogens was
evaluated by measuring cells viability and methanogenic activity of Methanospirillum
hungatei and Methanobacterium formicicum. The presence of hydrogenotrophic (M.
formicicum and M. hungatei) and acetoclastic (Methanosaeta concilii and
Methanosarcina. mazei) methanogens in oleate and palmitate enrichment cultures was
detected by PCR-DGGE fingerprinting techniques. Acetoclastic methanogens and M.
formicicum grew in oleate and palmitate enrichment cultures but M. hungatei only grew
in the palmitate’s enrichment. M. hungatei was more sensitive than M. formicicum
particularly to unsaturated LCFA. The later was also the most abundant hydrogenotroph
detected during the continuous treatment of a synthetic wastewater composed mainly
by oleate, as determined by PCR-DGGE. In the same study, M. concilii was identified as
the most representative acetoclast. In order to investigate differences between the
proteins expressed during the degradation of saturated and unsaturated LCFA, a
metaproteomics experiment was designed, in which an anaerobic sludge was incubated
with palmitate, stearate and oleate. The same COGs functional categories were
identified in the different conditions. The majority of the proteins were assigned to
functional categories, energy production and conversion, posttranslational modification
and lipid metabolism. Most of the proteins identified belong to Methanosaeta concilli,
Syntrophobacter fumaroxidans, Pelobacter propionicus and Pelotomaculum
thermopropionicum. Methanosaeta concilii was indeed the most abundant archaea
detected by pyrosequencing analysis of the 16S rRNA gene, but the other
microorganisms were not even detected by pyrosequencing. Studying metaproteomes
of complex microbial communities is still a big challenge especially because most of their
genomes are not sequenced which difficult proteins identification. Likewise, when
analyzing the proteome of the co-culture, Syntrophomonas zehnderi and M. formicicum,
specialized on the degradation of LCFA, M. formicicum’s proteome could be much better
characterized compared to S. zehnderi’s, since the genome of a very close related strain
of the former is available in public databases and the genome of S. zehnderi is not. S.
zehnderi was a dominant microorganism in oleate degrading enrichment cultures under
methanogenic and non-methanogenic conditions, stablishing close relationships with
hydrogenotrophic methanogens and homoacetogenic bacteria, respectively.Os ácidos gordos de cadeia longa (AGCL) podem ser convertidos a metano por digestão
anaeróbia. Contudo, aspectos relacionados com a microbiologia desta conversão ainda
não estão completamente compreendidos. Estudos anteriores sugerem que diferentes
microrganismos possam estar envolvidos na degradação de AGCL saturados e
insaturados e ainda que os AGCL inibem a actividade microbiológica e de forma mais
severa a actividade metanogénica. Neste trabalho foi avaliado o efeito toxico dos ACGL
saturados (palmitato (C16:0) e estearato (C18:0)) e insaturados (oleato (C18:1)) sobre
culturas puras de Methanobacterium formicicum e Methanospirillum hungatei
monitorizando a sua viabilidade celular e actividade metanogénica. A presença destes
organismos hidrogenotróficos bem como de dois organismos acetoclásticos,
Methanosaeta concilii e Methanosarcina mazei, em culturas mistas enriquecidas em
degradadores de palmitato e de oleato, foi determinada por PCR-DGGE. Todos
permaneceram nos dois enriquecimentos com excepção do M. hungatei que não foi
capaz de crescer no enriquecimento com oleato. Este microrganismo mostrou ser mais
sensível do que o M. formicicum aos AGCL insaturados, segundo os resultados de
viabilidade celular e actividade metanogénica. Em reactores anaeróbios alimentados
com um efluente sintético, composto maioritariamente por ácido oleico, M. formicicum
e M. concilii foram identificados como os metanogénicos predominantes. Foi efectuado
um estudo de metaproteómica com o objectivo de detectar diferenças na expressão de
proteínas por parte de uma cultura mista a crescer em AGCL saturados e insaturados. A
maioria das proteínas identificadas nos vários ensaios estava relacionada com processos
metabólicos de produção de energia, incluindo o metabolismo dos lípidos, e
modificação pós-traducional. A maioria das proteínas identificadas corresponde a
proteínas dos seguintes microrganismos: Methanosaeta concilli, Syntrophobacter
fumaroxidans, Pelobacter propionicus e Pelotomaculum thermopropionicum. Estas
amostras foram paralelamente classificadas taxonomicamente com base nos resultados
de pirosequenciação do gene que codifica para a subunidade 16S do rRNA. Segundo esta
análise a Methanosaeta concilii foi identificada como o organismo metanogénico mais
dominante, contudo, não foram detectadas sequências correspondentes a S.
fumaroxidans, P. propionicus ou P. thermopropionicum. A falta de informação
genética/proteica sobre os microrganismos envolvidos na degradação de AGCL dificulta
a identificação de proteínas que poderão ser relevantes neste processo. Analisou-se
também a expressão proteica de dois organismos sintróficos, Syntrophomonas zehnderi
and M. formicicum, que convertem os AGCL a metano. Neste caso particular foi possível
obter uma melhor caracterização do proteoma de M. formicicum do que de S. zehnderi,
consequência do facto de apenas o genoma de uma estirpe próxima do primeiro se
encontrar sequenciado. A S. zehnderi foi identificada como uma bactéria dominante em
culturas especializadas na degradação de oleato quer em condições metanogénicas,
estabelecendo relações de sintrofia com microorganismos metanogénicos
hidrogenotróficos, quer em condições não metanogénicas onde a interacção como
organismos homoacetogénicos terá sido favorecida.Research grant
(SFRH/BD/48960/2008) from the Portuguese Foundation for Science and
Technology (FCT) and European Social Fund (POPHQREN)Project
FCOMP-01-0124-FEDER-014784, financed by the FEDER funds through the
Operational Competitiveness Programme (COMPETE) and by national funds
through the Portuguese Foundation for Science and Technology (FCT
ABUNDANCE OF METHANOSAETA CONCILII-LIKE SPECIES IN THE SEDIMENT OF LEDBETTER CREEK EMBAYMENT OF KENTUCKY LAKE
No full textReservoirs, including Kentucky Lake, are unique in that they do not follow a natural cycle of rising during the rainy seasons and dropping during the dry summers. The lake is manipulated, mainly for flood control and transportation, so that the water level is increased during the summer months and decreased during the winter months. This manipulation leaves the littoral sediment of the lake submerged in the summer and exposed in the winter. The seasonal water level, along with other factors, like farming practices in the watershed, contributes to the availability of nutrients for organisms inhabiting the sediment of the lake. These littoral zone sediment, microbial communities are also not well understood. The Archaeal community of the Ledbetter Creek embayment, Kentucky Lake, has been the topic of study by several MSU students using sequence analysis of cloned 16S rDNA. BLAST searches showed species like the methanogen Methanosaeta concillii were the most abundant Archaea in this environment in 2007. Further, it was found that of the 59 M. concillii-like clones sequenced, 50 were unique species (using 98.7% sequence identity as the cutoff). The aim of this study was to again examine the Archaeal community in this environment to determine whether Methanosaeta concillii-like species continue to dominate the Archaeal community and to compare these sequences to those found in the previous study. DNA was extracted from sediment samples; the 16S rDNA genes were amplified using Archaeal specific primers and cloned. The cloned inserts were sequenced and compared to the Genebank database and the M. concilli-like sequences found previously. The results from this study show that the Methanosaeta concilli-like species remain abundant in these samples, but are not the same species found before, using the 98.7% identity cutoff. The results of this study also found that there is another group of archaea that were abundant in this environment, Methanoregula boonei-like organisms. These data suggest that the Archaeal community is continually in flux in that the organisms are very closely related to those found in 2007 and may be derivatives, but the species change with time
Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis
No full textCopper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease
Metaproteomics of anaerobic microbial communities degrading long-chain fatty acids
No full textThe anaerobic conversion of long-chain fatty acids (LCFA), and specifically the difference between the degradation of unsaturated- and saturated-LCFA, is not fully understood. In this work, syntrophic degradation of stearate (C18:0) and oleate (C18:1) was studied. A comparative metaproteomics approach, in which proteins were analyzed by LC-MS/MS, was combined with 16S rRNA gene pyrosequencing. Saturated- and unsaturated-LCFA were converted to methane by the anaerobic consortia. 16S rRNA gene pyrosequencing revealed differences in the microbial composition of sludges incubated with stearate and oleate, separately. Abundance of microorganisms within Deltaproteobacteria and within Synergistia taxa was higher in stearate and oleate incubations, respectively. Methanosaeta was the most abundant methanogen in both conditions. Metaproteomics results were similar and comparable distributions of COG functional categories were found for both samples. Archaeal proteomes were much better identified than bacterial ones, with five times more proteins retrieved. Most of the proteins identified belong to Methanosaeta concilli and Syntrophobacter fumaroxidans, two organisms that have their genome sequenced. Syntrophobacter belongs to Deltaproteobacteria, however this group was not dominant in oleate incubation as determined by pyrosequencing results. Studying metaproteomes of complex microbial communities is still a big challenge especially because most of the genomes are not sequenced which hinders protein identification
Identification of p38 MAPK and JNK as New Targets for Correction of Wilson Disease-Causing ATP7B Mutants
No full textWilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. Conclusion: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD
Activation of autophagy, observed in liver tissues from patients with Wilson disease and from Atp7b-deficient animals, protects hepatocytes from copper-induced apoptosis
No full textWilson disease is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in liver and brain. It is caused by mutations in the ATPase copper transporting beta gene (ATP7B), which encodes a protein that transports copper out of heaptocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to reduce copper toxicity in patients with Wilson disease
Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis
No full textGolgi-dependent copper homeostasis sustains synaptic development and mitochondrial content
No full textIndexación: Scopus.Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses. Copyright © 2021 the authors.https://www.jneurosci.org/content/41/2/215This work was supported by National Institutes of Health (NIH) Grants 1RF1AG060285 (to V.F.), R01NS108778 and R01NS108778-01S1 (to R.A.J.), R15AR070505 (to A.D.V.-M.), Telethon TIGEM-CBDM9 (to R.S.P.), R01NS086082 (to D.N.C.), R01GM083144 (to V.L.), and 5K12GM000680-19 (to C.H.). Stocks obtained from the Bloomington Drosophila Stock Center (NIH Grant P40OD018537) were used in this study.S
