Spin-trapping agent α-phenyl N-tert-butylnitrone binds to trypsin and enhances heparin-induced inhibition of amidolytic activity and structural degradation of the enzyme

AbstractThe effects of heparin on trypsin have recently been demonstrated to involve inhibition of catalytic activity and degradation of the enzyme by means of an oxidative mechanism. The possibility that α-phenyl N-tert-butylnitrone protects heparin-induced radical formation on trypsin was investigated by measuring amidolytic activity and changes in the structure of trypsin in the presence of heparin with and without α-phenyl N-tert-butylnitrone. The results show that α-phenyl N-tert-butylnitrone does not only prevent, but it even significantly enhances effects of heparin on the enzyme. This is due to the unique property of α-phenyl N-tert-butylnitrone, independently of spin-trapping capacity, to modify the trypsin structure by binding irreversibly to the catalytic triad, at sites distinct from those to which heparin binds

Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3′,4′,5′-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3′,4′,5′-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3′,4′,5′-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 μM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site

Biphasic pattern of heparin-induced oxidative degradation of trypsin in the presence of glucose.

The present work tested the hypothesis that the oxidative effects of heparin on trypsin may be modified by the addition of another reducing substance. Glucose was chosen as reducing sugar and used at a concentration (15 mM) and an incubation time (5 h at 37 degrees C) with trypsin which may cause only modest oxidative modifications, thus resembling what happens in vivo in the presence of slight, long-lasting hyperglycaemia. The effects of 10, 100 and 200 micrograms/mL standard heparin on both structural and functional properties of glucose-treated bovine trypsin were measured by UV and fluorescence emission spectra and by the proteolytic and esterolytic activities of trypsin on two different substrates. Radical formation was measured by the reduction of nitroblue tetrazolium and acetylated cytochrome c. The influence of incubation and desalting of solutions were also evaluated. In the absence of heparin but in the presence of glucose, no gross structural alteration of trypsin was observed, nor was any fragmentation of the enzyme visible in the electrophoretic pattern. However, despite the fact that soluble free radicals were not detectable, glucose did induce the formation of reactive species linked to trypsin itself, as confirmed by the findings of increased reduction of nitroblue tetrazolium by fresh undesalted, and incubated desalted glucose-treated trypsin solutions. Heparin, mostly at 10 micrograms/mL (which caused an increase of 9.64 nmol reduced cytochrome c/mg protein per min) significantly changed both the nature and concentration of the radicals which were formed. Heparin-induced oxygen radical production was rapid in onset, and markedly modified the structure and function of the enzyme which in time underwent complete inactivation and fragmentation. At both 100 and 200 micrograms/mL, heparin appeared to slow the oxidative process, as spectroscopic analysis and electrophoretic pattern showed less profound structural modifications of trypsin. Overall results show that heparin-induced oxidation of trypsin follows a biphasic, concentration-dependent pattern in the presence of another reducing substance such as glucose. Low heparin concentrations enhance the oxidative potential of glucose, whereas higher concentrations antagonize it

Complexes of Grp94 with human Immunoglobulin G

Complexes are described that form in vitro following incubation of "Heat Shock Protein" (HSP) "Glucose-regulated Protein" 94 (Grp94) with human non-immune immunoglobulin G. Results show that complexes of Grp94-lgG are resistant to denaturing agents. Moreover, complexes display an important cytokine-like property that can be exploited to induce positive effects of immuno-modulation in pathologies characterized by either a reduced or exacerbated immune response. In addition, stability of Grp94-lgG complexes make them useful as diagnostic tools to detect antibodies directed against Grp94 in various pathological conditions

Separation by heparin-affinity chromatography of catalytically active and inactive forms of trypsin which retain the (Na-K)ATPase stimulating property.

The possibility that different structural determinants on trypsin, other than catalytic sites, are involved in the cell membrane (Na-K)ATPase stimulating property was investigated by submitting bovine trypsin to two purification procedures: gel filtration on Sephadex G-50 and heparin-Sepharose chromatography. The latter procedure was also chosen in consideration of the known affinity for heparin displayed by serine proteinases. Trypsin peaks eluted from both columns were analysed by measuring esterolytic and proteolytic activities, the beef heart (Na-K)ATPase stimulating property and amino acid content. Fluorescence emission spectra and both non-denaturing and SDS-gel electrophoresis were also performed to test structural modifications on trypsin peaks. Four peaks eluted from Sephadex G-50 with variable estero-proteolytic and (Na-K)ATPase stimulating activities; the latter was also present in two peaks which displayed the lowest estero-proteolytic activities. All peaks proved to be trypsin in amino acid composition. Two peaks eluted from the heparin-Sepharose column with distinct biological activities: a first minor peak, eluted with the void volume, was catalytically inactive but it retained the (Na-K)ATPase stimulating activity. The second, major peak eluted mostly with 0.5 mol/l NaCl, displayed only esteroproteolytic activities, but no (Na-K)ATPase-stimulating activity. It overlapped control trypsin in both electrophoretic patterns, fluorescence emission spectrum and amino acid composition. The first peak showed differences with the parent compound, as revealed by the amino acid composition and tryptophan fluorescence emission spectrum. Marked differences were also observed in the electrophoretic pattern which only showed bands of low molecular mass mostly confined to the anode. NH2-terminus analysis confirmed that the first peak contained trypsin fragments originated from the parent compound after passage through the heparin column. It is hypothesized that trypsin binding to heparin causes structural alteration of the proteinase and primes the catalytic cleavage of fragments which lose heparin affinity and elute in the void volume. The results also confirm that the proteolytic mechanism is not involved in trypsin-mediated (Na-K)ATPase stimulation and indicate that heparin-Sepharose chromatography is a useful tool to separate catalytically active and inactive forms of trypsin

The role played by serine proteases in the development and worsening of vascular complications in Type 1 diabetes mellitus.

Much attention has been given to the role played by serine proteases in the development and worsening of vascular complications in Type 1 diabetes mellitus. A generalized increase in proteolytic activity, either due to a true increase in concentration of specific proteases or defects of their protease inhibitors, represents an early marker of diabetes. However, the precise molecular mechanism whereby an unopposed proteolytic activity leads to overt vascular alterations has not fully been elucidated as yet. The picture is further complicated by the fact that, although sharing the same function, serine proteases constitute a structurally heterogeneous class of molecules. Besides classical proteases, for most part belonging to coagulative and fibrinolytic systems, other unrelated molecules exhibit serine-like protease activity and are capable of triggering both inflammatory and immune reactions. The specific role of these non classical serine proteases in the complex pathogenesis of diabetes and its vascular complications is attracting a new investigative interest, as these molecules may represent additional therapeutic targets. This review will focus on most recent acquisitions on this issue relevant to Type 1 diabetes

Albumin contamination of a purified human alpha1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor.

A partially purified preparation of human alpha 1-antitrypsin (alpha 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of alpha 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed alpha 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of alpha 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with beta-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the alpha 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from alpha 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the alpha 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose alpha 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed alpha 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified alpha 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of alpha 1-AT. Results indicate that the mobility of alpha 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the alpha 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity