RESUMO
The characterization of adeno-associated virus (AAV)-based gene therapy products represents significant challenges owing to their extremely large molecular sizes, structural complexity and heterogeneity, and limited sample amounts. Mass spectrometry (MS) is one of the key analytical tools that can overcome these challenges and serve as an important technique for the analysis of multiple attributes. In this review, the current methodologies and emerging trends in MS analysis of AAV gene therapy products are presented, highlighting their advantages and unique capabilities in addressing key issues encountered in intact AAV vector analysis, capsid viral protein characterization and impurity analysis.
Assuntos
Proteínas do Capsídeo , Dependovirus , Dependovirus/genética , Dependovirus/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Terapia Genética , Espectrometria de Massas , Vetores GenéticosRESUMO
Transcription factor DksA contains a four-Cys Zn(2 +)-finger motif thought to be responsible for structural integrity and the relative disposition of its domains. Pseudomonas aeruginosa encodes an additional DksA paralog (DksA2) that is expressed selectively under Zn(2+) limitation. Although DksA2 does not bind Zn(2+), it complements the Escherichia coli dksA deletion and has similar effects on transcription in vitro. In this study, structural and biochemical analyses reveal that DksA2 has a similar fold, domain structure and RNA polymerase binding properties to those of the E. coli DksA despite the lack of the stabilizing metal ion.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Pseudomonas aeruginosa/química , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Transcrição Gênica , Zinco/química , Zinco/metabolismoRESUMO
We recently described a novel GnRH receptor signaling pathway mediated by the prostaglandins (PGs) F(2alpha) and PGI(2), which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and inhibit LH but not FSH release. Here we further explore the cross talk between GnRH and the PG receptors. GnRH stimulates arachidonic acid (AA) release from LbetaT2 gonadotrope cells via the Ca(2+)-independent phospholipase A(2) (iPLA(2)) and not via the more common Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha). AA release was followed by a marked induction of cyclooxygenase (COX)-1 and COX-2 by GnRH via the protein kinase C/c-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription by GnRH is mediated by the two nuclear factor-kappaB sites and the CCAAT/enhancer-binding protein site within its promoter. Indeed, GnRH stimulates p65/RelA phosphorylation (22-fold) in LbetaT2 cells and the two nuclear factor-kappaB sites apparently act as a composite response element. Although GnRH stimulates cAMP formation in LbetaT2 cells, we found no role for cAMP acting via the cAMP response element site in the COX-2 promoter. PGF(2alpha), PGI(2), or PGE(2) had no effect on GnRH-stimulated ERK, c-Jun N-terminal kinase, and p38MAPK activation or on GnRH- and high K(+)-stimulated intracellular Ca(2+) elevation in LbetaT2 and gonadotropes in primary culture. Although, PGF(2alpha), PGI(2), and PGE(2) reduced GnRH-stimulated cAMP formation, we could not correlate it to the inhibition of GnRH receptor expression, which is exerted only by PGF(2alpha) and PGI(2.) Hence, the inhibition by PGF(2alpha) and PGI(2) of the autoregulation of GnRH receptor expression is most likely mediated via inhibition of GnRH-stimulated phosphoinositide turnover and not by inhibition of Ca(2+) elevation and MAPK activation.