Huntingtonova choroba (HD) je autozomálně dominantní neurodegenerativní onemocnění způsobené zvýšením počtu polyglutaminových repetic (> 35 repetic) v genu pro protein huntingtin. HD je charakteristická pomalými progresivními změnami pohybového aparátu a osobnosti, kdy tyto změny jsou často doprovázeny ztrátou tělesné hmotnosti. Do dnešního dne není znám přesný mechanizmus patofyziologie choroby. Poruchy pohybových funkcí reflektují masivní poškození specifických částí mozku (striatum), které bylo popsáno u pacientů s HD. V roce 2013 Sbodio et al [1] popsali zvýšené množství proteinu Acyl‑CoA binding domain containing 3 (ACBD3) ve striatu HD pacientů. Protein ACBD3 hraje nezastupitelnou roli v mnoha buněčných procesech, a to především díky interakci s různými vazebnými partnery. ACBD3 je esenciální při neuronálním dělení, neurodegeneraci, udržení lipidové homeostáze, stresové odpovědi, virové replikaci, apoptóze, udržení struktury golgiho komplexu. V této práci jsme prokázali nepřítomnost proteinu ACBD3 v mitochondriích v lidských kožních fibroblastech a navíc jsme potvrdili, že změny celkové hladiny proteinu ACBD3 ve fibroblastech HD pacientů nejsou konzistentní.

Huntington's disease (HD) is an autosomal‑dominant neurodegenerative disease caused by the expansion of polyglutamine repeats (> 35 repeats) in the nuclear gene for the huntingtin protein. HD is characterized by slow progressive changes in motor behaviour and personality that are sometimes accompanied by weight loss. To date, the exact mechanisms of HD pathophysiology have not been defined. Impaired motor behaviour reflecting massive and selective destruction of the striatum has been observed in patients with HD. Sbodio et al. [1] reported in 2013 that Acyl‑CoA binding domain containing 3 (ACBD3) protein levels were elevated in the striatum of HD patients and connected with higher neurotoxicity in HD. The ACBD3 protein plays essential roles in many different cellular functions via interactions with a multitude of partners. ACBD3 is involved in neuronal stem cell self‑renewal, neurodegeneration, lipid homeostasis, stress resistance, intracellular vesicle trafficking, organelle maintenance, viral replication and the apoptotic response. Herein, we found that ACBD3 in not present in the mitochondria in skin fibroblasts. Moreover, our findings also revealed that the total cellular level of ACBD3 is not consistent among the fibroblasts of HD patients.

• Klíčová slova
• lidské kožní fibroblasty, Acyl-CoA binding domain containing 3 protein,
• MeSH
• adaptorové proteiny signální transdukční * fyziologie   metabolismus   MeSH
• buněčné linie metabolismus   MeSH
• dospělí MeSH
• fibroblasty metabolismus   MeSH
• frakcionace buněk MeSH
• heterozygot MeSH
• Huntingtonova nemoc * metabolismus   MeSH
• imunohistochemie MeSH
• kůže * metabolismus   patologie   MeSH
• lidé MeSH
• membránové proteiny * fyziologie   metabolismus   MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• interakce hostitele a patogenu * MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Picornaviridae fyziologie   MeSH
• replikace viru * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• vazba proteinů MeSH
• virové proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• MeSH
• acylkoenzym A chemie   metabolismus   MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• Picornaviridae chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   metabolismus   MeSH
• Golgiho aparát metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• biologický transport fyziologie   MeSH
• ceramidy metabolismus   MeSH
• cholesterol metabolismus   MeSH
• glykosylace MeSH
• Golgiho aparát metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• membránový protein 2 asociovaný s lyzozomy metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• transferasy pro jiné substituované fosfátové skupiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Picornaviruses are small positive-sense single-stranded RNA viruses that include many important human pathogens. Within the host cell, they replicate at specific replication sites called replication organelles. To create this membrane platform, they hijack several host factors including the acyl-CoA-binding domain-containing protein-3 (ACBD3). Here, we present a structural characterization of the molecular complexes formed by the non-structural 3A proteins from two species of the Kobuvirus genus of the Picornaviridae family and the 3A-binding domain of the host ACBD3 protein. Specifically, we present a series of crystal structures as well as a molecular dynamics simulation of the 3A:ACBD3 complex at the membrane, which reveals that the viral 3A proteins act as molecular harnesses to enslave the ACBD3 protein leading to its stabilization at target membranes. Our data provide a structural rationale for understanding how these viral-host protein complexes assemble at the atomic level and identify new potential targets for antiviral therapies.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   metabolismus   MeSH
• aminokyselinové motivy MeSH
• buněčné linie MeSH
• exprese genu MeSH
• interakce hostitele a patogenu * MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• Kobuvirus genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• replikace viru genetika   MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• unilamelární lipozómy chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virové nestrukturální proteiny chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Picornaviruses infect a wide range of mammals including livestock such as cattle and swine. As with other picornavirus genera such as Aphthovirus, there is emerging evidence of a significant economic impact of livestock infections caused by members of the genera Enterovirus and Kobuvirus. While the human-infecting enteroviruses and kobuviruses have been intensively studied during the past decades in great detail, research on livestock-infecting viruses has been mostly limited to the genomic characterization of the viral strains identified worldwide. Here, we extend our previous studies of the structure and function of the complexes composed of the non-structural 3A proteins of human-infecting enteroviruses and kobuviruses and the host ACBD3 protein and present a structural and functional characterization of the complexes of the following livestock-infecting picornaviruses: bovine enteroviruses EV-E and EV-F, porcine enterovirus EV-G, and porcine kobuvirus AiV-C. We present a series of crystal structures of these complexes and demonstrate the role of these complexes in facilitation of viral replication.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• bovinní enterovirus patogenita   MeSH
• buněčné linie MeSH
• enterovirové infekce metabolismus   veterinární   virologie   MeSH
• enteroviry prasat genetika   patogenita   MeSH
• HEK293 buňky MeSH
• Kobuvirus genetika   patogenita   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• pikornavirové infekce metabolismus   veterinární   virologie   MeSH
• prasata MeSH
• replikace viru genetika   MeSH
• skot MeSH
• virové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• maloúhlový rozptyl MeSH
• membránové proteiny metabolismus   MeSH
• multimerizace proteinu * MeSH
• proteiny 14-3-3 metabolismus   MeSH
• rab proteiny vázající GTP metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. OBJECTIVE: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. METHODS: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. RESULTS: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. CONCLUSIONS: The gradual development of a neurodegenerative phenotype, ac-companied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

• MeSH
• fenotyp MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc genetika   MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• mozek metabolismus   MeSH
• prasata MeSH
• protein huntingtin genetika   MeSH
• proteiny nervové tkáně genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH