Disrupting proteasomal and autophagic degradation systems of misfolded alpha-sarcoglycan protein by bortezomib and givinostat combination

Background and Purpose: Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. Experimental Approach: We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Key Results: Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Conclusion and Implications: Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.

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Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F

Gene ◽

10.1016/j.gene.2006.02.007 ◽

2006 ◽

Vol 376 (1) ◽

pp. 59-67 ◽

Cited By ~ 36

Author(s):

Sandra Morales-Arrieta ◽

Maria Elena Rodríguez ◽

Lorenzo Segovia ◽

Agustín López-Munguía ◽

Clarita Olvera-Carranza

Keyword(s):

Leuconostoc Mesenteroides ◽

Functional Characterization ◽

Gene Encoding

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Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽

10.3390/molecules23112876 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2876 ◽

Cited By ~ 2

Author(s):

Lin Tan ◽

Mei Wang ◽

Youfa Kang ◽

Farrukh Azeem ◽

Zhaoxi Zhou ◽

...

Keyword(s):

Enzyme Assay ◽

Molecular Mechanisms ◽

Mangifera Indica ◽

Functional Characterization ◽

Citrate Buffer ◽

Anthocyanidin Reductase ◽

High Amino Acid ◽

Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

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Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

mBio ◽

10.1128/mbio.01877-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 24

Author(s):

Travis J. Wiles ◽

Elena S. Wall ◽

Brandon H. Schlomann ◽

Edouard A. Hay ◽

Raghuveer Parthasarathy ◽

...

Keyword(s):

Comparative Study ◽

Molecular Mechanisms ◽

Genetic Manipulation ◽

Functional Characterization ◽

Symbiotic Bacteria ◽

Bacterial Isolates ◽

Major Barrier ◽

Allelic Exchange ◽

Genetic Techniques

ABSTRACTCorrelating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis andin vitroassays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCEA great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

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Cloning and functional characterization of the gene encoding the transcription factor Acel in the basidiomycete Phanerochaete chrysosporium

Biological Research ◽

10.4067/s0716-97602006000500007 ◽

2006 ◽

Vol 39 (4) ◽

Cited By ~ 6

Author(s):

RUBÉN POLANCO ◽

PAULO CANESSA ◽

ALEXIS RIVAS ◽

LUIS F LARRONDO ◽

SERGIO LOBOS ◽

...

Keyword(s):

Transcription Factor ◽

Phanerochaete Chrysosporium ◽

Functional Characterization ◽

Gene Encoding

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Functional characterization of the Haemophilus influenzae 4.5S RNA

Canadian Journal of Microbiology ◽

10.1139/w97-124 ◽

1998 ◽

Vol 44 (1) ◽

pp. 91-94

Author(s):

G Scott Jenkins ◽

Mark S Chandler ◽

Pamela S Fink

Keyword(s):

Haemophilus Influenzae ◽

Functional Characterization ◽

Coli Strain ◽

Northern Analysis ◽

Gene Encoding ◽

E Coli ◽

Functional Homolog ◽

Polymerase Chain ◽

4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

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Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein�1 in hot pepper (Capsicum annuum L. cv. Pukang)

Planta ◽

10.1007/s00425-004-1412-5 ◽

2004 ◽

Vol 220 (6) ◽

pp. 875-888 ◽

Cited By ~ 77

Author(s):

Jong-Pil Hong ◽

Woo Taek Kim

Keyword(s):

Capsicum Annuum ◽

Functional Characterization ◽

Hot Pepper ◽

Gene Encoding ◽

Capsicum Annuum L ◽

Binding Factor ◽

Responsive Element

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Characterization of an N-terminal Nav1.5 channel variant – a potential risk factor for arrhythmias and sudden death?

BMC Medical Genetics ◽

10.1186/s12881-020-01170-3 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Stefanie Scheiper-Welling ◽

Paolo Zuccolini ◽

Oliver Rauh ◽

Britt-Maria Beckmann ◽

Christof Geisen ◽

...

Keyword(s):

Sudden Death ◽

Functional Characterization ◽

Missense Variant ◽

Hek293 Cells ◽

Patch Clamp Technique ◽

Gene Encoding ◽

Heterozygous Variant ◽

Cardiac Sodium Channel ◽

Electrophysiological Measurements

Abstract Background Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. Methods Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. Results Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. Conclusion The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

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Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Blood ◽

10.1182/blood-2002-05-1397 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3626-3632 ◽

Cited By ~ 161

Author(s):

Barbara Plaimauer ◽

Klaus Zimmermann ◽

Dirk Völkel ◽

Gerhard Antoine ◽

Randolf Kerschbaumer ◽

...

Keyword(s):

Von Willebrand Factor ◽

Functional Activity ◽

Functional Characterization ◽

Eukaryotic Expression ◽

Gene Encoding ◽

Hek 293 ◽

293 Cells ◽

Von Willebrand ◽

Willebrand Factor

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designatedADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.

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Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

Applied and Environmental Microbiology ◽

10.1128/aem.00599-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7104-7112 ◽

Cited By ~ 59

Author(s):

Maria Karczmarczyk ◽

Yvonne Abbott ◽

Ciara Walsh ◽

Nola Leonard ◽

Séamus Fanning

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Gene Array ◽

Multidrug Resistant ◽

Genetic Determinants ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

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