Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Molecular biology of plasma membrane redox enzymes: A survey of current knowledge

Summary Despite a large body of evidence for enzymatic activities and physiological functions of plasma membrane redox function, few of these enzymes have been characterized in terms of molecular biology. Examples for these with at least some molecular data up to complete sequences, membrane topology and binding sites for substrates and coenzymes or prosthetic groups are NADH-ferricyanide reductase of Ehrlich ascites membranes, NADH-coenzyme Q reductase of liver, NADH oxidase ectoenzyme of liver and HeLa (and possibly other) cells, protein disulfide isomerase which is widespread, and relatives thereof, as well as cytochromes P-450 andb ₅₅₈, NADPH oxidase of fat and thyroid cells and fat cell amine oxidase. Ferricyanide reductase and coenzyme O reductase may be identical, but NADH oxidase ectoenzyme is distinct and possibly functions also as a disulfide and a copper reductase. On the other hand, the plasma-membrane-located protein disulfide isomerase (PDI), despite its similar enzymatic activity, is completely different from the ectooxidase. The latter is shed from the membrane into the surrounding medium by proteolysis, whereas PDI is not an integral membrane protein and is secreted intact. Another disulfide reductase has been demonstrated in THP-1 cells, which again is totally different from the former two. It turns out that enzymatic activities are insufficient to describe redox enzymes. Special forms of cytochrome P-450 can be induced to expression at the cell membrane of liver, where they are transported by the cytoskeleton-associated secretory pathway. Why some isoforms are expressed at the surface and some are not is not yet clear. Cytochromeb ₅₅₈, the flavocytochrome of neutrophils, is described in other cells too, but there are different isoforms, which are genetically distinct. A relative has also been identified in duodenal cells, where it functions as a ferric reductase involved in iron absorption. NADPH oxidase of fat cells has very similar properties, but the identity is unproved, whereas thyroid oxidase is a non-heme protein which is calcium-sensitive and does not need assembly of subunits for activation. Finally, fat cell membranes also possess a quinone-containing amine-oxidase which may be involved in signaling of glucose-transport regulation, as it is also found in GLUT4-containing vesicles. However, the physiological connection has yet to be demonstrated.     

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.     

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.     

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.     

Retarded protein folding of the human Z-type α1-antitrypsin variant is suppressed by Cpr2p

The human Z-type α₁-antitrypsin variant has a strong tendency to accumulate folding intermediates due to extremely slow protein folding within the endoplasmic reticulum (ER) of hepatocytes. Human α₁-antitrypsin has 17 peptidyl-prolyl bonds per molecule; thus, the effect of peptidyl-prolyl isomerases on Z-type α₁-antitrypsin protein folding was analyzed in this study. The protein level of Cpr2p, a yeast ER peptidyl-prolyl isomerase, increased more than two-fold in Z-type α₁-antitrypsin-expressing yeast cells compared to that in wild-type α₁-antitrypsin-expressing cells. When CPR2 was deleted from the yeast genome, the cytotoxicity of Z-type α₁-antitrypsin increased significantly. The interaction between Z-type α₁-antitrypsin and Cpr2p was confirmed by co-immunoprecipitation. In vitro folding assays showed that Cpr2p facilitated Z-type α₁-antitrypsin folding into the native state. Furthermore, Cpr2p overexpression significantly increased the extracellular secretion of Z-type α₁-antitrypsin. Our results indicate that ER peptidyl-prolyl isomerases may rescue Z-type α₁-antitrypsin molecules from retarded folding and eventually relieve clinical symptoms caused by this pathological α₁-antitrypsin.     

Population structure of Spodoptera frugiperda maize and rice host forms in South America: are they host strains?

Determining which factors contribute to the formation and maintenance of genetic divergence to evaluate their relative importance as a cause of biological differentiation is among the major challenges in evolutionary biology. In Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) two host strains have been recognized in the 1980s: the corn‐strain prefers maize, sorghum, and cotton, whereas the rice‐strain prefers rice and wild grasses. However, it is not clear to what extent these so‐called ‘strains’, which have also been called ‘host races’ or even ‘sibling species’, are really associated with host plants. Due to the indeterminate evolutionary status, we will use the term ‘host forms’ (sensu Funk). Here, we characterized populations collected from maize, rice, and wild grasses from three countries in South America. Using two mitochondrial cytochrome oxidase I (mtCOI) markers and 10 polymorphisms in the triose phosphate isomerase (Tpi) gene, we found various patterns of host association. Two hundred twenty‐seven nuclear amplified fragment length polymorphisms (AFLPs) markers revealed significant genetic differentiation among populations, which was generally correlated to the host from which the larvae were collected. Using a multivariate discriminant analysis and a Bayesian clustering approach, we found that individuals could be grouped into 2–5 genetically distinct clusters, depending on the method. Together, our results indicate that although host‐associated differentiation is present in this species, it does not account for all observable genetic variation and other factors must be maintaining genetic differentiation between these forms. Therefore, the term ‘host strains’ should be abandoned and ‘host forms’ should be used instead for S. frugiperda.     

Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

Lipopolysaccharide (LPS) biosynthesis represents an underexploited target pathway for novel antimicrobial development to combat the emergence of multidrug‐resistant bacteria. A key player in LPS synthesis is the enzyme D ‐arabinose‐5‐phosphate isomerase (API), which catalyzes the reversible isomerization of D ‐ribulose‐5‐phosphate to D ‐arabinose‐5‐phosphate, a precursor of 3‐deoxy‐D ‐manno‐octulosonate that is an essential residue of the LPS inner core. API is composed of two main domains: an N‐terminal sugar isomerase domain (SIS) and a pair of cystathionine‐β‐synthase domains of unknown function. As the three‐dimensional structure of an enzyme is a prerequisite for the rational development of novel inhibitors, we present here the crystal structure of the SIS domain of a catalytic mutant (K59A) of E. coli D ‐arabinose‐5‐phosphate isomerase at 2.6‐Å resolution. Our structural analyses and comparisons made with other SIS domains highlight several potentially important active site residues. In particular, the crystal structure allowed us to identify a previously unpredicted His residue (H88) located at the mouth of the active site cavity as a possible catalytic residue. On the basis of such structural data, subsequently supported by biochemical and mutational experiments, we confirm the catalytic role of H88, which appears to be a generally conserved residue among two‐domain isomerases.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.