Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.     

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.     

CODON OPTIMIZATION INCREASES HUMAN KALLISTATIN EXPRESSION IN Escherichia coli

A unique serpin, kallistatin, displays vasodilatory, antiangiogenic, anti-inflammatory, and antioxidant activity. Difficulty and low efficacy of obtaining recombinant kallistatin limit the wide investigation of its biological and pathological function. The present study employed a codon optimization algorithm to redesign the kallistatin gene and achieved a high yield of recombinant kallistatin protein. The kallistatin codons were redesigned for a more suitable Escherichia coli host without altering amino acids. Base composition and GC% content were compared between synthetic optimized kallistatin (opti-kallistatin) and wild-type kallistatin (wt-kallistatin). Both opti-kallistatin and wt-kallistatin were purified using Ni-NTA His-binding resins through fast protein liquid chromatography (FPLC). The identity and purity of kallistatin were confirmed by Coomassie blue staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis. The output of opti-kallistatin protein was ～2-fold increase (2.09 ± 0.23 mg/L) compared to wt-kallistatin (1.05 ± 0.2 mg/L). These results suggest that more common codon optimization in the E. coli host significantly increases the yield of heterologous human protein yields. This approach will remarkably facilitate the further investigation of kallistatin in vitro and in vivo.     

MiR-3691-5p is upregulated in docosahexaenoic acid-treated vascular endothelial cell and targets serpin family E member 1

Endothelium, the inner cellular lining of blood vessels, has an important role in the regulation of physiological processes and its dysfunction may initiate cardiovascular complications. Previous investigations have revealed that dietary docosahexaenoic acid (DHA) is related to a lower possibility of cardiovascular disease and mortality. Until now, the molecular mechanisms in the biological activities of DHA remain largely unknown. MicroRNAs (miRNAs) play a vital role in regulating gene expression. Thus, we aimed to investigate whether DHA improves the dysfunction via regulating miRNAs. To understand the protective effects of DHA through modulating miR-3691-5p and its target genes for palmitic acid (PAL) induced apoptosis in endothelial cells. The present study demonstrated that DHA upregulated miR-3691-5p expression, and downregulated the expression of its target gene serpin family E member 1 (SERPINE1). MiR-mimics and inhibitors modulation results indicated that miR-3691-5p regulates endothelial apoptosis through activating antiapoptotic response which controlled by the STAT3 signaling pathway. In conclusion, we have shown that PAL-induced apoptosis could be decreased by DHA treatment through miR-3691-5/SERPINE1 pathways.     

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Acid Denaturation of alpha1-antitrypsin: characterization of a novel mechanism of serpin polymerization

The native serpin architecture is extremely sensitive to mutation and environmental factors. These factors induce the formation of a partially folded species that results in the production of inactive loop-sheet polymers. The deposition of these aggregates in tissue, results in diseases such as liver cirrhosis, thrombosis, angioedema and dementia. In this study, we characterize the kinetics and conformational changes of alpha(1)-antitrypsin polymerization at pH 4 using tryptophan fluorescence, circular dichroism, turbidity changes and thioflavin T binding. These biophysical techniques have demonstrated that polymerization begins with a reversible conformational change that results in partial loss of secondary structure and distortion at the top of beta-sheet A. This is followed by two bimolecular processes. First, protodimers are formed, which can be dissociated by changing the pH back to 8. Then, an irreversible conformational change occurs, resulting in the stabilization of the dimers with a concomitant increase in beta-sheet structure, allowing for subsequent polymer extension. Electron microscopy analysis of the polymers, coupled with the far-UV CD and thioflavin T properties of the pH 4 polymers suggest they do not form via the classical loop-beta-sheet A linkage. However, they more closely resemble those formed by the pathological variant M(malton). Taken together, these data describe a novel kinetic mechanism of serine proteinase inhibitor polymerization.     

The activation of the atypical PKC zeta in light‐induced retinal degeneration and its involvement in L‐DNase II control

Light‐induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase‐independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI‐derived DNase II (LEI/L‐DNase II), a caspase‐independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro‐survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L‐DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase‐independent pathways.     

Probing the local conformational change of alpha1-antitrypsin

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.     

An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: Application to neuroserpin reveals regions that differentially regulate inhibitory activity

The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution‐matrix‐based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface‐exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site‐directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins. Proteins 2015; 83:135–152. © 2014 Wiley Periodicals, Inc.