Lisa A. Collins-Racie

[21] Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli

Publisher Summary In this chapter, protocols are provided for the use of thioredoxin gene fusion expression system. It describes a variety of suitable E. coli expression strains and a number of thioredoxin expression vectors. It also presents the procedures for thioredoxin fusion protein purification and method for specific cleavage of thioredoxin fusions by enterokinase. Thioredoxin fusions have proved to be especially useful in avoiding inclusion body formation, particularly for the production of small, normally secreted, mammalian cytokines in an active form in the E. coli cytoplasm. E. coli thioredoxin is a compact, highly soluble, and thermally stable protein with robust folding characteristics. These properties perhaps allow the molecule, when fused to a protein of interest, to serve as a covalently joined molecular chaperon. Thioredoxin may, thus, act to prevent the aggregation and precipitation of fused nascent proteins, giving them an extended opportunity to adopt their correct tertiary folds. Thioredoxin also possesses a number of additional characteristics that suit it for the role as a fusion partner. It is small, highly translated, and its tertiary structure reveals that both its amino and carboxyl termini are accessible for potential fusions to other molecules. Moreover, its active site comprises a surface-accessible loop that can be utilized for internal peptide insertions. Purifications of thioredoxin fusion proteins can be facilitated by making use of the remarkable ability of the molecule to be released from the bacterial cytoplasm by simple osmotic shock, by taking advantage of the inherent thermal stability of the molecule, by using avidin or streptavidin matrices to capture thioredoxin variants modified to allow for in vivo biotinylation, or by using the engineered forms of thioredoxin with affinity to metal chelate column matrices.

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor‐bound enzymes exhibit two essentially different catalytic‐site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

Measurement of myostatin concentrations in human serum: Circulating concentrations in young and older men and effects of testosterone administration ☆

UNLABELLED Methodological problems, including binding of myostatin to plasma proteins and cross-reactivity of assay reagents with other proteins, have confounded myostatin measurements. Here we describe development of an accurate assay for measuring myostatin concentrations in humans. Monoclonal antibodies that bind to distinct regions of myostatin served as capture and detector antibodies in a sandwich ELISA that used acid treatment to dissociate myostatin from binding proteins. Serum from myostatin-deficient Belgian Blue cattle was used as matrix and recombinant human myostatin as standard. The quantitative range was 0.15-37.50 ng/mL. Intra- and inter-assay CVs in low, mid, and high range were 4.1%, 4.7%, and 7.2%, and 3.9%, 1.6%, and 5.2%, respectively. Myostatin protein was undetectable in sera of Belgian Blue cattle and myostatin knockout mice. Recovery in spiked sera approximated 100%. ActRIIB-Fc or anti-myostatin antibody MYO-029 had no effect on myostatin measurements when assayed at pH 2.5. Myostatin levels were higher in young than older men (mean+/-S.E.M. 8.0+/-0.3 ng/mL vs. 7.0+/-0.4 ng/mL, P=0.03). In men treated with graded doses of testosterone, myostatin levels were significantly higher on day 56 than baseline in both young and older men; changes in myostatin levels were significantly correlated with changes in total and free testosterone in young men. Myostatin levels were not significantly associated with lean body mass in either young or older men. CONCLUSION Myostatin ELISA has the characteristics of a valid assay: nearly 100% recovery, excellent precision, accuracy, and sufficient sensitivity to enable measurement of myostatin concentrations in men and women.

Protein Kinase Cζ Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-κB by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes

Protein kinase Cζ (PKCζ) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCζ is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCζ may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCζ, implicating PKCζ activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCζ activity in NF-κB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-α caused an increase in NF-κB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCζ-sparing inhibitor calphostin C. A cell-permeable PKCζ pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-κB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCζ protein effectively prevented cytokine-mediated NF-κB activation in primary chondrocytes. These data implicate PKCζ as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.

We have isolated a zebrafish cadherin that is orthologous to human LI-cadherin (CDH17). Zebrafish cdh17 is expressed exclusively in the pronephric ducts during embryogenesis, and in the mesonephros during larval development and adulthood. Like its mammalian ortholog, cdh17 is also expressed in liver and intestine in adult zebrafish. We show that cdh17-positive mesodermal cells do not contribute to the hematopoietic system. Consistent with a cell adhesion role for Cdh17, depletion of Cdh17 function using antisense morpholino oligonucleotides compromised cell cohesion during pronephric duct formation. Our results indicate that Cdh17 is necessary for maintaining the integrity of the pronephric ducts during zebrafish embryogenesis. This finding contrasts with the role of mammalian CDH17, which does not appear to be involved in nephric development.

LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E2 (PGE2) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRα and -β) are expressed in cartilage, with LXRβ being the predominant isoform. Here we show that genetic disruption of Lxrβ gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE2 production in articular cartilage treated with IL-1β, indicating a protective role of LXRβ in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE2 production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE2 synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

Global analysis of nuclear receptor expression and dysregulation in human osteoarthritic articular cartilage: Reduced LXR signaling contributes to catabolic metabolism typical of osteoarthritis

OBJECTIVE Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. METHOD The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. RESULTS Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRalpha and LXRbeta, and their heterodimeric partners retinoid X receptor (RXR)alpha and RXRbeta were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. CONCLUSIONS Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18–24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole‐mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5′ primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.

Cloning and expression of a novel cysteine-rich secreted protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo

We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.