Protein and Peptide Letters - Volume 20, Issue 12, 2013

Toxoplasma gondii is ubiquitous obligate intracellular parasite and is one of the most important pathogen for humans and animals. In humans, T. gondii has two life forms: tachyzoites and bradyzoites. Tachyzoites form of T. gondii can cause acute infection, and it is called toxoplasmosis. The development of latent bradyzoites from rapidly growing tachyzoites has been linked to cellular and environmental stresses which are associated with heat shock proteins (Hsps). Hsps play a protective role against stressors. Hsp40 is an important member of Hsp family and T. gondii has 36 predicted Hsp40 family members. Therefore, we studied the cloning and biochemical characterization of the T. gondii RH strain Hsp40 protein-Gok1. Hsp40 prevents protein aggregation and induce refolding. Consequently, Hsp40s may play essential roles in the mechanisms of bradyzoite development and survival in the host organism. Hsp40-Gok1 functional and structural properties may facilitate drug design and protein targeting against toxoplasmosis.

L-asparaginases (E.C.3.5.1.1, L- ASNases) have been widely used in clinical practice as chemotherapeutic drugs of acute lymphoblastic leukaemia (ALL). In order to evaluate the structural and functional role of selected residues in ASNases we report the screening of a library of L-ASNase mutants aiming to find detrimental mutations that significantly affect catalysis and substrate specificity. The library of mutants was created using the staggered extension process (StEp) and the genes of L- ASNases from Erwinia chrysanthemi (ErL-ASNase) and Erwinia carotovora (EcaL-ASNase). A mutant that displayed dramatic reduction in L-asparaginase activity and undetectable activity towards L-Gln and Nα - acetyl-L-Asn was isolated and characterized. Sequence of the mutant showed that it has a single point aminoacid replacement (Gly281Ser) of the E. carotovora enzyme. Steady-state kinetic analysis demonstrated that the Gly281Ser aminoacid replacement influence significantly the enzyme’s structural and functional properties. In particular it displays 10.8-fold increase in Km and 45.5-fold lower catalytic activity towards L-Asn. Analysis of the pH dependence of Vmax and Vmax/Km of L-Asn hydrolysis showed that the mutant displays modified properties regarding the dependence of kinetic constants towards the pH. Studies on the thermal stability of the mutant enzyme demonstrated that it exhibits about 3.8°C higher halfinactivation temperature, compared to the wild-type enzyme, suggesting that Gly281 contributes to the low stability of the enzyme. Biocomputing analysis suggested that the Gly281Ser replacement causes indirectly changes in the active site architecture and presumably alters the dynamics of the enzyme.

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or β-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.

Recombinant technology has ushered in a new era for the pharmaceutical industry. Protein therapeutics, including plasma-derived products and antibodies obtained from the serum of infected patients, have been successfully adopted and utilized to treat various indications. The development of recombinant technology and the subsequent improvement in expression, purification, and formulation technologies have enabled the generation of highly purified proteins in a scalable and cost-effective manner. The discovery and development of several recombinant proteins, such as growth factors and cytokines, will be described followed by a brief review of monoclonal antibodies and enzyme replacement therapy. Recombinant protein-based vaccine, which is the focus of the current review, is described in detail with particular emphasis on several viral and bacterial infections. Challenges and new approaches in their use as a replacement for the currently available vaccines are discussed.

The identification of novel molecular targets has paved the way for new treatment options in cancer patients. A number of agents targeting molecules that are crucial both for the tumor and its microenvironment have already been approved by the U.S. Food and Drug Administration for clinical use. The monoclonal antibodies and the small molecule kinase inhibitors constitute two major classes of targeted therapeutic agents, which have apparently different mechanisms of action, toxicity profiles, routes of administration, timing and dosing. Moreover, individual differences in genes regulating the distribution and metabolism of targeted agents evidently influence treatment outcomes. Data regarding the immune- and tumor microenvironment-modulatory properties of most of these agents are either obscure or controversial, as well. Therefore, preclinical animal and human studies that aim to identify the immunological, biological and the pharmacological properties of these novel classes of agents that also employ recent developments in pharmacogenomics and proteomics are warranted.

UDP-glucuronosyltransferase (UDP) catalyzes a broad spectrum of endobiotic and xenobiotics. It is the isoform of the UGT1 family, which are made from the complex gene locus by an alternative combination of one of the unique first exons with the commonly used exons. UDP is believed to be involved in cellular activity and signaling process associated with detoxification. In this study, rats were orally administered with 18β-glycyrrhetinic acid (GA) for four weeks before rats were sacrificed. The mRNA was extracted from the liver and cDNA was prepared by reverse transcription method. By PCR and Northern blot analysis, UGT1A8 mRNA level was found to be increased in the treatment group (P < 0.05). The results showed that the mRNA expression of UGT1A8 could be induced both by GA and the licorice extract. GA was identified to be the active component of the aqueous extract of licorice responsible for induction of UGT1A8 in the rat liver. The results suggest a transcriptional control of the UGT1A8 synthesis can be modulated by phytochemicals in the rat liver. The increased UDP activity could enhance detoxification of toxicants.

In recent years, therapeutic proteins have become an important growing class of drugs in the pharmaceutics industry. The development of recombinant DNA technology has caused to appreciation of therapeutic value of many proteins and peptides in medicine. Currently, approximately 100 therapeutic proteins obtained approval from Food and Drug Administration (FDA) and they are widely used in the treatment of various diseases such as cancer, diabetes, anemia and infections. This paper will summarize the production processes, pharmaceuticals and physicochemical properties and important classes of therapeutic proteins with their potential use in clinical applications.

Over the past 20 years the demand for recombinant proteins has increased significantly. Mammalian cell lines have been extensively used to produce recombinant proteins. This expression system offers several advantages over microbial systems, mammalian cells have the cellular machinery to promote the secretion of the recombinant product and the posttranslational modifications, like glycosylation that is present in many of recombinant therapeutic proteins in the market. Human cell lines have emerged as a new and powerful alternative for production of such products. These cells are able to produce recombinant proteins with posttranslational modifications more similar to their natural counterparts, producing proteins with human-like glycosylation pattern avoiding immunogenic reactions against epitopes nonhumans. This review presents the available human cell lines that can be used in pharmaceutical industry, the advantages of this expression system and the main efforts made in this field.

Homocysteine (Hcys) is a non-essential amino acid associated with a range of diseased and abnormal metabolic conditions. Hcys concentration in saliva is routinely determined by enzyme assays, which are broadly specific, but can be expensive and suffer from cross-reactivity. Total Hcys (tHcys) concentrations in eight healthy adults were determined to establish the inter-day variation during resting, normal and intensive physical activity, using the more sensitive analytical techniques of liquid chromatography and tandem mass spectrometry without prior derivatization. Saliva (~ 1.5 mL) was collected over four days; early morning (EA), normal activity (NA) and during physical activity (PA). Samples were processed by disulphide reduction, acetonitrile precipitation and then centrifugation-filtration. Extracts were chromatographically resolved and analysed on a quadrupole time- of-flight (QToF) mass spectrometer. The protonated [M+H]+, m/z 136.101 and product ([M+H]+- HCOOH) m/z 90.103 ions were then monitored against an internal standard (13CHcys) and external set of calibration standards. Mean tHcys concentration for the whole group, including exercise was 6.6 ± 8.0 (range 0.2 – 29.6 nmol/mL). Overall, concentration of tHcys was greater in males than the females but not significantly (p > 0.05). The mean EA concentration was significantly < 0.05) greater than NA for both males (p = 0340) and females (p = 0.0045). There were large within-subject variations (coefficient of variation; CV%; 24% to 103%). The limits of detection (LOD) and quantification (LOQ) were 0.07 and 0.22 nmol/mL, respectively. The procedure potentially provides a convenient means of analyzing salivary Hcys as a diagnostic disease marker.