A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

Endostatin in Renal and Cardiovascular Diseases

Endostatin, a protein derived from the cleavage of collagen XVIII by the action of proteases, is an endogenous inhibitor known for its ability to inhibit proliferation and migration of endothelial cells, angiogenesis, and tumor growth. Angiogenesis is defined as the formation of new blood vessels from pre-existing vasculature, which is crucial in many physiological processes, such as embryogenesis, tissue regeneration, and neoplasia. <b><i>Summary:</i></b> Increasing evidence shows that dysregulation of angiogenesis is crucial for the pathogenesis of renal and cardiovascular diseases. Endostatin plays a pivotal role in the regulation of angiogenesis. Recent studies have provided evidence that circulating endostatin increases significantly in patients with kidney and heart failure and may also contribute to disease progression. <b><i>Key Message:</i></b> In the current review, we summarize the latest findings on preclinical and clinical studies analyzing the impact of endostatin on renal and cardiovascular diseases.

CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action

CXCL17 is the most recently described chemokine. It is principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

The F1Fo-ATPase inhibitor, IF1, is a critical regulator of energy metabolism in cancer cells

In the last two decades, IF1, the endogenous inhibitor of the mitochondrial F1Fo-ATPase (ATP synthase) has assumed greater and ever greater interest since it has been found to be overexpressed in many cancers. At present, several findings indicate that IF1 is capable of playing a central role in cancer cells by promoting metabolic reprogramming, proliferation and resistance to cell death. However, the mechanism(s) at the basis of this pro-oncogenic action of IF1 remains elusive. Here, we recall the main features of the mechanism of the action of IF1 when the ATP synthase works in reverse, and discuss the experimental evidence that support its relevance in cancer cells. In particular, a clear pro-oncogenic action of IF1 is to avoid wasting of ATP when cancer cells are exposed to anoxia or near anoxia conditions, therefore favoring cell survival and tumor growth. However, more recently, various papers have described IF1 as an inhibitor of the ATP synthase when it is working physiologically (i.e. synthethizing ATP), and therefore reprogramming cell metabolism to aerobic glycolysis. In contrast, other studies excluded IF1 as an inhibitor of ATP synthase under normoxia, providing the basis for a hot debate. This review focuses on the role of IF1 as a modulator of the ATP synthase in normoxic cancer cells with the awareness that the knowledge of the molecular action of IF1 on the ATP synthase is crucial in unravelling the molecular mechanism(s) responsible for the pro-oncogenic role of IF1 in cancer and in developing related anticancer strategies.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.