From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>

Disulfide bonds formed between a pair of oxidized cysteines are important to the structural integrity and proper folding of many proteins. Accordingly, Nature has evolved several systems for the genesis and maintenance of such bonds. Beginning with the discovery of protein disulfide isomerase, which provided the first evidence for enzyme-catalyzed disulfide-bond formation, many years of research have resulted in the explication of the complex network of electron transport pathways needed for this process. Herein, we take a historical approach in describing the elucidation of disulfide-bond formation in E. coli. We frame this topic in the context of genome sequencing eras. The first section describes the discovery of eukaryotic protein disulfide isomerase and the subsequent research that followed from the early 1960s to the early 1990s, a time period we have named the pre-genomic sequencing era. The second section details the renaissance in research on disulfide-bond formation in the periplasm of prokaryotes, fueled by bacterial genetic screens and the development of genomic sequencing technology. Accordingly, we have named this section the genomic sequencing era, which ranges from the early 1990s to approximately 2010. The final section outlines the use of bacterial genetic screens to select for new oxidoreductase enzymes and their potential uses in biotechnological and pharmaceutical applications. This era we have dubbed the post-genomic sequencing era, and we envision it to represent the future of research on oxidative folding

Identification of a novel putative interaction partner of the nucleoporin ALADIN

It has been shown that the nucleoporin ALADIN plays a significant role in the redox homeostasis of the cell, but its function in steroidogenesis contributing to adrenal atrophy in triple A syndrome remains largely unknown. In an attempt to identify new interaction partners of ALADIN, co-immunoprecipitation followed by proteome analysis was conducted in different expression models using the human adrenocortical tumour cell line NCI-H295R. Our results suggest an interaction of ALADIN with the microsomal protein PGRMC2. PGRMC2 is shown to be activity regulator of CYP P450 enzymes and, therefore, to be a possible target for adrenal dysregulation in triple A syndrome. We show that there is a sexual dimorphism regarding the expression of Pgrmc2 in adrenals and gonads of wild-type (WT) and Aaas knock-out (KO) mice. Female Aaas KO mice are sterile due to delayed oocyte maturation and meiotic spindle assembly. A participation in meiotic spindle assembly confirms the recently investigated involvement of ALADIN in mitosis and emphasises an interaction with PGRMC2 which is a regulator of the cell cycle. By identification of a novel interaction partner of ALADIN, we provide novel aspects for future research of the function of ALADIN during cell cycle and for new insights into the pathogenesis of triple A syndrome

Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

Background: Membrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxification. PGRMC1 and PGRMC2 belong to the membrane-associated progesterone receptor family and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other; thereby suppressing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic controller Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. Results: In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over-expressed. Furthermore, we find that depletion of ALADIN results in mislocalization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fibroblasts. Conclusion: Our results emphasize the possibility that loss of the regulatory association between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibers. This observation may explain part of the symptoms seen in triple A syndrome patients.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non‐syndromic form of isolated familial achalasia

Background Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease. Methods Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study. Key Results A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol:IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP(3)R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entireMRVI1gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases withMRVI1mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform beta (cGK1 beta) depicted a partial mislocalization of cGK1 beta due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1 beta. Conclusions & Inferences In individuals with an early onset of achalasia without further accompanying symptoms,MRVI1mutations should be considered as the disease-causing defect

Anne Frank and After

Between 1940 and 1945, 110,000 of the 140,000 Dutch Jews were deported to the death camps in Eastern Europe. 80% never returned. In Anne Frank and After the authors focus on two main questions: how exactly did this happen, and how has Dutch literature come to terms with this appalling event? In the book's final chapter they analyze the relationship between history and the literature of the Holocaust. Does literature add to what we know or does it actually distort historical evidence? Based on the work of leading historians of the period, the book examines literary works from Gerard Durlacher, Anne Frank, W.F. Hermans, Harry Mulisch, Gerard Reve and many others

Compensation for chronic oxidative stress in ALADIN null mice

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response

Regulation of CREB Phosphorylation in Nucleus Accumbens after Relief Conditioning

Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKII&alpha;, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning

convertibleCARs: A chimeric antigen receptor system for flexible control of activity and antigen targeting.

We have developed a chimeric antigen receptor (CAR) platform that functions as a modular system to address limitations of traditional CAR therapies. An inert form of the human NKG2D extracellular domain (iNKG2D) was engineered as the ectodomain of the CAR to generate convertibleCARTM-T cells. These cells were specifically directed to kill antigen-expressing target cells only in the presence of an activating bispecific adapter comprised of an iNKG2D-exclusive ULBP2-based ligand fused to an antigen-targeting antibody (MicAbodyTM). Efficacy against Raji tumors in NSG mice was dependent upon doses of both a rituximab-based MicAbody and convertibleCAR-T cells. We have also demonstrated that the exclusive ligand-receptor partnering enabled the targeted delivery of a mutant form of IL-2 to selectively promote the expansion of convertibleCAR-T cells in vitro and in vivo. By altering the Fv domains of the MicAbody or the payload fused to the orthogonal ligand, convertibleCAR-T cells can be readily targeted or regulated