Alterations in the proportions of skeletal muscle proteins following a unilateral lesion to the substantia nigra pars compacta of rats

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30–70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of α-tropomyosin and increased the proportion of β-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T_1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson’s disease may be associated with alterations to the expression of skeletal muscle proteins.

Identification of two proteins, S14 and UIP1, that interact with UCH37

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targetsof reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Subcellular localisation of proteins in fluorescent microscope images using a random forest

This paper presents a system that employs random forests to formulate a method for subcellular localisation of proteins. A random forest is an ensemble learner that grows classification trees. Each tree produces a classification decision, and an integrated output is calculated. The system classifies the protein-localisation patterns within fluorescent microscope images. 2D images of HeLa cells that include all major classes of subcellular structures, and the associated feature set are used. The performance of the developed system is compared against that of the support vector machine and decision tree approaches. Three experiments are performed to study the influence of the training and test set size on the performance of the examined methods. The calculated classification errors and execution times are presented and discussed. The lowest classification error (2.9%) has been produced by the developed system.

A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.

Generation of mutant leukaemia inhibitory factor (LIF)-IgG heavy chain fusion proteins as bivalent antagonists of LIF

Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel–nitrilotriacetic acid (Ni–NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.

Whey protein and gum arabic encapsulated Omega-3 lipids. The effect of material properties on coacervation

The effect of material properties on complex coacervation of whey protein and gum Arabic from various sources was investigated. In this study, it was demonstrated that material properties of whey protein isolates and gum Arabic affect the complex coacervation process significantly. For whey protein, the coacervation capability could be correlated with their level of denaturation and calcium content. For gum Arabic, both material sources and salt content were found to be attributing factors to their coacervation capability. This study facilitated the development of Omega-3 lipids microcapsules with promising performances in certain food applications.