BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

• Klíčová slova
• Hepatocellular carcinoma, Metallothionein-3, Resistance, Sorafenib,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• ACBD3, RNA virus, coarse-grained simulations, host factor, intrinsically disordered regions, picornavirus, small-angle X-ray scattering (SAXS),
• 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
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• acylkoenzym A MeSH
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH

Diaporthe species are important pathogens, saprobes, and endophytes on grapevines. Several species are known, either as agents of pre- or post-harvest infections, as causal agents of many relevant diseases, including swelling arm, trunk cankers, leaf spots, root and fruit rots, wilts, and cane bleaching. A growing body of evidence exists that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation, during plant development and responses to biotic and abiotic stresses. In this study, we explored differentially expressed miRNAs in response to Diaporthe eres and Diaporthe bohemiae infection in Vitis vinifera cv. Chardonnay under in vitro conditions. We used computational methods to predict putative miRNA targets in order to explore the involvement of possible pathogen response pathways. We identified 136 known and 41 new miRNA sequence variants, likely generated through post-transcriptional modifications. In the Diaporthe eres treatment, 61 known and 17 new miRNAs were identified while in the Diaporthe bohemiae treatment, 101 known and 21 new miRNAs were revealed. Our results contribute to further understanding the role miRNAs play during plant pathogenesis, which is possibly crucial in understanding disease symptom development in grapevines infected by D. eres and D. bohemiae.

• Klíčová slova
• RT-qPCR, grapevine, high-throughput sequencing, miRNA,
• MeSH
• Ascomycota patogenita   MeSH
• mikro RNA genetika   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• transkriptom MeSH
• Vitis genetika   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA 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
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Rab proteiny vázající GTP MeSH
• rab11 protein MeSH Prohlížeč
• rekombinantní proteiny MeSH

Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• Kobuvirus enzymologie   MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• pikornavirové infekce metabolismus   virologie   MeSH
• virové nestrukturální proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfatidylinositolfosfáty MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• virové nestrukturální proteiny 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
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfatidylinositolfosfáty MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• phosphatidylinositol 4-phosphate MeSH Prohlížeč