Paola Finotti

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

Alteration of plasma proteinase-antiproteinase system in type 1 diabetic patients. Influence of sex and relationship with metabolic control

The aim of the present study was to investigate whether or not alterations of the plasma proteinase-antiproteinase system were present in type 1 (insulin-dependent) diabetic patients and, if so, whether or not they were related to sex, age at onset and duration of the disease as well as to short- and long-term diabetic control. The plasma concentration of trypsin-like activity and two of the most important plasma serine proteinase inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin, were determined in 95 type 1 diabetic and 67 control subjects. The plasma concentration of alpha 1 antitrypsin was found to be markedly decreased (P < 0.001), whereas plasma alpha 2-macroglobulin and trypsin-like activity were increased in diabetics compared to controls (P = 0.009 and < 0.001, respectively). Sex also influenced the values of both proteinase inhibitors in diabetics, women showing higher values of plasma alpha 1-antitrypsin (P = 0.004) than men. In women, HbA1c was also positively correlated with blood glucose (P < 0.001), daily insulin dosage (P < 0.001), and trypsin-like activity of plasma (P = 0.02). On the contrary, in men, HbA1c appeared to be negatively correlated with plasma alpha 2-macroglobulin (P = 0.02). In addition to sex, age at onset (but not duration) of the disease revealed differences in plasma alpha 1-antitrypsin among diabetics, the lowest mean value of this inhibitor being present in men with age at onset below 15 years, who also showed a significant negative correlation between this inhibitor and HbA1c (P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Structure–Activity Relationship in a Purine-Scaffold Compound Series with Selectivity for the Endoplasmic Reticulum Hsp90 Paralog Grp94

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.

Stable complexes formed by Grp94 with human IgG promoting angiogenic differentiation of HUVECs by a cytokine-like mechanism.

To explore the molecular mechanisms by which complexes of Grp94 with IgG, purified from the plasma of diabetic subjects, could drive an inflammatory risk in vascular cells, native Grp94 was co-incubated with human, non-immune IgG to obtain the formation of complexes that were then tested on human umbilical vein endothelial cells (HUVECs). Co-incubation of Grp94 with IgG led to the formation of stable, SDS-resistant complexes that displayed effects partly similar and partly significantly different from those of Grp94 alone. Both Grp94 alone and with IgG stimulated the cell growth and promoted angiogenesis by a mechanism of autocrine/paracrine activation of the expression of heat shock protein (HSP)90 and HSP70. However, the most striking alterations in the cell cytoskeleton, characterized by dramatic rearrangement of actin and increased formation of podosomes, were induced by Grp94 with IgG, and were mediated by the enhanced expression of HSP90. At variance with Grp94 alone, Grp94 with IgG promoted the angiogenic differentiation by activating a signaling pathway apparently independent of the intense stimulation of the ERK1/2 pathway that was instead more directly involved in mediating the proliferative effects on HUVECs. Results show unprecedented cytokine-like effects of Grp94 and a so far undisclosed capacity to bind irreversibly IgG, forming complexes that, with respect to Grp94 alone, display a more intense angiogenic transforming capacity that may predict an increased inflammatory risk in vascular cells in vivo.

Albumin contamination of a purified human α1-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.

Heparin-induced structural and functional alterations of bovine trypsin

To investigate the mechanism whereby heparin can modulate the activity of serine proteinases, bovine trypsin was chosen as reference and treated with heparin at 10, 100 and 200 micrograms/ml, in buffer solvents, with and without incubation at 37 degrees C. Heparin caused rapid, buffer- and pH-dependent decrease in trypsin solubility due to the generation of insoluble fragments from proteinase. Desalting treatments variously restored solubility by removing insoluble material. UV absorption and fluorescence emission spectra revealed significant heparin-induced conformational alterations in the trypsin molecule, the maximal effect being apparent at a proteinase-to-heparin molar ratio ranging from 1.6 to 1.0. The involvement of the catalytic sites of trypsin by heparin was further confirmed by the significant reduction in the difference absorption spectra of proflavine. Both proteolytic and esterolytic activities of trypsin were shown to be markedly decreased by heparin, especially after 5 h incubation at 37 degrees C. However, when the proteolytic and esterolytic activities of trypsin were measured on fresh solutions not submitted to desalting treatments, variable activation instead of inhibition of both activities was observed in the presence of heparin, this effect waning spontaneously in time or after desalting treatment. The paradoxical increase in functional activities was not inhibited by soybean trypsin inhibitor and was accompanied by denaturation and fragmentation of the proteinase as demonstrated by spectroscopic analyses and SDS-PAGE of fresh solutions. The results obtained indicated that heparin causes a rapid, time- and temperature-dependent conformational alteration of trypsin with irreversible denaturation and degradation of the proteinase. The underlying mechanism appears to be heparin-catalyzed oxidative degradation of trypsin due to liberation of oxygen radicals which are also responsible for the temporary increase in catalytic functions.

Identification and partial purification of a (Na-K)ATPase stimulating serine protease from plasma of insulin-dependent diabetics

Plasma from insulin-dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 microgram/ml of thrombin, 0.50 microgram/ml of kallikrein and 0.55 microgram/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 micrograms/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.

Angiogenic transforming capacity of IgG purified from plasma of type 1 diabetic patients

We previously demonstrated that plasma of type 1 diabetic patients contains antibodies complexed irreversibly with Grp94 that also display proteolytic activity. In this work, we wanted to test whether antibodies obtained from diabetic plasma may convey an inflammatory risk on vascular cells. To this aim, IgG were purified on the Protein‐G column from individual plasma of eight type 1 diabetic patients, and then tested on HUVECs to measure effects on cell growth and morphologic changes at different incubation times. The purified fractions of IgG contained a significant amount of Fab/(Fab)2, both free and in big aggregates, and anti‐Grp94 antibodies, mostly irreversibly linked with, but also free of Grp94. The purified fractions of both Fab/(Fab)2 and whole IgG stimulated the proliferation and sustained the angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) at sub‐nanomolar concentrations. IgG from normal plasma neither stimulated the cell growth nor induced any differentiation of HUVECs. The maximum cell growth stimulation occurred at 6–9 hrs and associated with the strong activation of the ERK1/2 pathway, whereas angiogenic transformation was completed later when the ERK1/2 activation was silenced and cell growth stimulation significantly reduced. Neither proteolytic activity of MMP‐9 nor VEGF were apparently involved in mediating the angiogenic differentiation of HUVECs that mostly correlated with an increased expression of HSP70 closely coupled with cell membrane‐bound inactive species of MMP‐9. Results indicate that effects displayed on HUVECs by antibodies purified from diabetic plasma are likely sustained by immune complexes with Grp94 that may thus predict an increased risk of angiogenic transformation in vivo.

Mechanism of Na+/K+-ATPase activation by trypsin and kallikrein

The mechanism of the Na+/K(+)-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K(+)-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 microgram/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 micrograms/ml. Na+/K(+)-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na(+)-ATPase activity was stimulated to a greater extent (up to 350%), whereas K(+)-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K(+)-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K(+)-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K(+)- or Na(+)-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K(+)-ATPase at site(s) located on the extracellular moiety of the enzyme.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.