Sequence similarity of HSP65 of Mycobacterium bovis BCG with SARS-CoV-2 spike and nuclear proteins: may it predict an antigen-dependent immune protection of BCG against COVID-19?

The Bacillus Calmette-Guérin (BCG) vaccine is known to have protective effects not only against tuberculosis but also against other unrelated infectious diseases caused by different pathogens. Several epidemiological studies have also documented the beneficial influence of BCG vaccine in reducing both susceptibility to and severity of SARS-CoV-2 infection. The protective, non-specific effects of BCG vaccination would be related to an antigen-independent enhancement of the innate immunity, termed trained immunity. However, the knowledge that heat shock protein (HSP)65 is the main antigen of Mycobacterium bovis BCG prompted us to verify whether sequence similarity existed between HSP65 and SARS-CoV-2 spike (S) and nuclear (N) proteins that could support an antigen-driven immune protection of BCG vaccine. The results of the in silico investigation showed an extensive sequence similarity of HSP65 with both the viral proteins, especially SARS-CoV-2 S, that also involved the regions comprising immunodominant epitopes. The finding that the predicted B cell and CD4+ T cell epitopes of HSP65 shared strong similarity with the predicted B and T cell epitopes of both SARS-CoV-2 S and N would support the possibility of a cross-immune reaction of HSP65 of BCG with SARS-CoV-2.

A mutant α1antitrypsin in complex with heat shock proteins as the primary antigen in type 1 diabetes in silico investigation.

Based on previous results demonstrating that complexes of a mutant α1-antitrypsin with the heat shock proteins (HSP)70 and glucose-regulated protein94 (Grp94) circulate in the blood of patients with type 1 diabetes, we raised the hypothesis that these complexes could represent the primary antigen capable of triggering the autoimmune reactions leading to overt diabetes. As a first approach to this issue, we searched whether A1AT and HSPs had a sequence similarity to major islet antigen proteins so as to identify among the similar sequences those with potential relevance for the pathogenesis of diabetes. A thorough in silico analysis was performed to establish the score of similarity of the human proteins: A1AT, pro-insulin (INS), GAD65, IAPP, IA-2, ICA69, Grp94, HSP70 and HSP60. The sequences of A1AT and HSPs with the highest score of similarity to the islet peptides reported in the literature as the main autoantigens in human diabetes were recorded. At variance with other HSPs, also including HSP90 and Grp78, Grp94 contained the highest number and the longest sequences with structural similarity to A1AT and to well-known immunogenic peptides/epitopes of INS, GAD65, and IA-2. The similarity of A1AT with Grp94 and that of Grp94 with INS also suggested a functional relationship among the proteins. Specific sequences were identified in A1AT, Grp94 and HSP70, with the highest score of cross-similarity to a pattern of eight different islet protein epitopes. The similarity also involved recently discovered autoantigens in type 1 diabetes such as a hybrid peptides of insulin and the defective ribosomal insulin gene product. The significant similarity displayed by specific sequences of Grp94 and A1AT to the islet peptides considered main antigens in human diabetes, is a strong indication for testing these sequences as new peptides of immunogenic relevance in diabetes.

Effects of purine-scaffold inhibitors on HUVECs: Involvement of the purinergic pathway and interference with ATP. Implications for preventing the adverse effects of extracellular Grp94.

BACKGROUND: Extracellular Glucose-regulated protein94 (Grp94) is linked to pathological conditions disrupting the obligatory intracellular location of this Heat Shock Protein (HSP). In plasma, Grp94 is linked to IgG in complexes that drive adverse effects on vascular cells and are biomarker of gastro-intestinal cancer. By blocking ATP site in different HSPs, purine-scaffold inhibitors are used as promising anti-cancer compounds, but their effects on vasculature are not known. METHODS: We tested the capacity of two purine-scaffold inhibitors, PU-H71 and PU-WS13, to prevent the binding of Grp94 to IgG and to antagonize the effects of Grp94 and native Grp94-IgG complexes on HUVECs in different experimental conditions. RESULTS: PU-H71 and PU-WS13 blocked Grp94 and the formation of Grp94-IgG complexes in absence of cells. Instead, in presence of HUVECs rather than Grp94 PU-inhibitors targeted cells causing stimulation of Akt and VEGF pathways and displaying angiogenic-like effects similar to, although less intense than that provoked by Grp94 and Grp94-IgG complexes. Unlike Grp94 and Grp94-IgG complexes, PU-inhibitors also activated the purinergic pathway and increased the expression of the ATP receptor P2X7. Effects of PU-inhibitors on HUVECs were reversed by ATP and in presence of ATP PU-inhibitors were again able to block Grp94. CONCLUSIONS: PU-inhibitors can display direct effects on endothelial cells by targeting the ATP receptor P2X7. In absence of ATP, PU-inhibitors preferentially bind to cells rather than Grp94. ATP antagonizes the PU-inhibitor binding to cells thus restoring the capacity to block Grp94 and Grp94-IgG complex formation. Results have implications for enhancing the therapeutic efficacy of PU-inhibitors against circulating pathogenic Grp94.

Grp94 in complexes with IgG is a soluble diagnostic marker of gastrointestinal tumors and displays immune-stimulating activity on peripheral blood immune cells.

Glucose-regulated protein94 (Grp94), the most represented endoplasmic reticulum (ER)-resident heat shock protein (HSP), is a tumor antigen shared by different types of solid and hematological tumors. The tumor-specific feature of Grp94 is its translocation from the ER to the cell surface where it displays pro-oncogenic functions. This un-physiological location has important implications for both the tumor pathology and anti-tumor therapy. We wanted to address the question of whether Grp94 could be measured as liquid marker in cancer patients in order to make predictions of diagnostic and therapeutic relevance for the tumor. To this aim, we performed an in-depth investigation on patients with primary tumors of the gastrointestinal (GI) tract, using different methodological approaches to detect Grp94 in tumor tissues, plasma and peripheral blood mononuclear cells (PBMCs). Results indicate that Grp94 is not only the antigen highly expressed in any tumor tissue and in cells of tumor infiltrates, mostly B lymphocytes, but it is also found in the circulation. However, the only form in which Grp94 was detected in the plasma of any patients and in B lymphocytes induced to proliferate, was that of stable complexes with Immunoglobulin (Ig)G. Using a specific immune-enzyme assay to measure plasma Grp94-IgG complexes, we showed that Grp94-IgG complexes were significantly increased in cancer patients compared to healthy control subjects, serving as diagnostic tumor biomarker. Results also demonstrate that the stimulation of patient PBMCs with Grp94-IgG complexes led to an increased secretion of inflammatory cytokines that might drive a potentially beneficial anti-tumor effect.

Human IgGs induce synthesis and secretion of IgGs and neonatal Fc receptor in human umbilical vein endothelial cells.

Human IgGs are increasingly used in the therapy of many different immune and inflammatory diseases, however their mechanism of action still remains unclear in most diseases. To gain insight into the mechanism by which IgGs might also exert their effects on endothelial cells, we tested human IgGs on human umbilical vein endothelial cells (HUVECs). IgGs induced a time-dependent increase in the synthesis and secretion of IgGs, together with a marked angiogenic-like transformation of HUVECs that was maximal after a 20-h incubation. IgGs stimulated IG gene transcription without affecting the process of gene rearrangement, already present in control HUVECs. The mechanism involved the activation of transcription factors with the increased expression of HSP90, HSP70 and inactive MMP-9 responsible for the phenotypic differentiation associated with the most intense IgG synthesis and secretion. However, even a short incubation with IgGs followed by recovery of cells was sufficient to trigger and sustain in time the synthesis and secretion of new IgGs, independently of the angiogenic-like transformation visible only when cells were continuously exposed to IgGs. Under the stimulus of IgGs, specific secretory pathways were also activated in HUVECs together with the expression of FcRn, which was always associated with IgGs of new synthesis, forming complexes that were also secreted. Our results disclose a so far unknown and unexpected mechanism of IgGs on HUVECs that behave as Ig-producing immune cells. Results might have relevance for the effects that IgGs also exert in vivo in physiological conditions.

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.

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/ß 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.

Structural insights into complexes of glucose-regulated Protein94 (Grp94) with human immunoglobulin G. relevance for Grp94-IgG complexes that form in vivo in pathological conditions.

While the mechanism by which Grp94 displays its chaperone function with client peptides in the cell has been elucidated extensively, much less is known about the nature and properties of how Grp94 can engage binding to proteins once it is exposed on the cell surface or liberated in the extra-cellular milieu, as occurs in pathological conditions. In this work, we wanted to investigate the molecular aspects and structural characteristics of complexes that Grp94 forms with human IgG, posing the attention on the influence that glycosylation of Grp94 might have on the binding capacity to IgG, and on the identification of sites involved in the binding. To this aim, we employed both native, fully glycosylated and partially glycosylated Grp94, and recombinant, non-glycosylated Grp94, as well as IgG subunits, in different experimental conditions, including the physiological setting of human plasma. Regardless of the species and type, Grp94 engages a similar, highly specific and stable binding with IgG that involves sites located in the N-terminal domain of Grp94 and the hinge region of whole IgG. Grp94 does not form stable complex with Fab, F(ab)2 or Fc. Glycosylation turns out to be an obstacle to the Grp94 binding to IgG, although this negative effect can be counteracted by ATP and spontaneously also disappears in time in a physiological setting of incubation. ATP does not affect at all the binding capacity of non-glycosylated Grp94. However, complexes that native, partially glycosylated Grp94 forms with IgG in the presence of ATP show strikingly different characteristics with respect to those formed in absence of ATP. Results have relevance for the mechanism regulating the formation of stable Grp94-IgG complexes in vivo, in the pathological conditions associated with the extra-cellular location of Grp94.

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

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90ß, 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.

Inhibition of immunoglobulin secretion from peripheral blood mononuclear cells by glucose-regulated protein94 (Grp94) in allergic subjects.

Glucose-regulated protein94 (Grp94) is the most represented endoplasmic reticulum-resident HSP with the unique property to modulate the immune response. This has opened the way to the use of Grp94 as effective therapeutic agent in both depressed and exaggerated activity of the immune system. We investigated the effect of native Grp94 on peripheral blood mononuclear cells (PBMCs) isolated from blood of two subjects with a different history of bronchial allergic asthma. Whereas in subject 1 an elevated basal level of Ig and altered morphological aspects of PBMCs suggested an intense antigen-driven stimulation of the immune system, subject 2 had an apparently normal basal humoral response. However, Grp94 reduced in a concentration-dependent manner the Ig secretion from PBMCs of both subjects, inhibition being maximal at 100 ng/ml Grp94 after 15 days. The effect was apparently related to inhibition of intra-cellular content of both IgG and IgE, and in subject 1 was still observed a year after the first examination. Dot-blot experiments revealed the presence of anti-Grp94 antibodies in Ig secreted from PBMCs and in plasma of both subjects, confirming the role of Grp94 as antigen responsible for activation of the immune system, even in the absence of clinical signs of asthma. Anti-Grp94 antibodies significantly decreased after PBMC treatment with Grp94 at 100 ng/ml. Results demonstrate that inhibition of the humoral response by Grp94 crucially depends on being Grp94, the antigen challenging the immune system in these allergic subjects, thus supporting the role of Grp94 as immuno-modulatory agent in pathologies with exaggerated immune response.

Crucial role of HSP90 in the Akt-dependent promotion of angiogenic-like effect of glucose-regulated protein94 (Grp94)-IgG complexes.

Previous observations showed that complexes of glucose-regulated protein94 (Grp94) with human IgG, both those isolated from plasma of diabetic subjects and complexes formed in vitro, displayed cytokine-like effects on human umbilical vein endothelial cells (HUVECs), including angiogenic-like transformation capacity that predicted an increased risk of vascular damage. The aim of the present work was to find an effective inhibitor of the angiogenic-like effect of Grp94-IgG complexes. Because this effect is mediated by an increased expression of matrix metalloprotease-9 (MMP-9), we tested the selective MMP-9 inhibitor, the cyclic decapeptide CTT (CTTHWGFTLC) at 5, 10 and 20 µM. CCT failed to inhibit any morphological alteration induced by Grp94-IgG on HUVECs, on its own displaying a paradoxical angiogenic-like activity. We identified the phosphatidylinositol 3-kinase (PI3K)/Akt pathway as the specific target activated by both Grp94-IgG and CTT for sustaining the angiogenic-like transformation of HUVECs. Functioning of the PI3K/Akt pathway was crucially dependent on functional heat-shock protein (HSP)90, and both Grp94-IgG and CTT caused and increased expression of HSP90, promoting its localization to podosomes. CTT appeared to enhance the angiogenic-like effect of Grp94-IgG by increasing the rate of secretion of both HSP90 and MMP-9. By preventing the chaperoning capacity of HSP90 with the inhibitor purine-scaffold (PU)-H71 that blocked the ATP-binding site on HSP90, it was possible to inhibit the expression of Akt and secretion of HSP90 and MMP-9 induced by Grp94-IgG, thus completely reversing the angiogenic pattern. Results reveal a fundamental role of HSP90 in the PI3K/Akt pathway-mediated angiogenic-like effect of Grp94-IgG, also questioning the capacity of CTT to serve as an effective inhibitor of the angiogenic effect.

Effects of glucose-regulated protein94 (Grp94) on Ig secretion from human blood mononuclear cells.

Grp94 is the main endoplasmic reticulum-resident heat shock protein (HSP) that besides chaperoning native proteins, displays important modulatory effects on both the innate and adaptive immune response. Since the knowledge of a direct influence of Grp94 on the humoral response is lacking, in this work we tested the effect of Grp94 on Ig secretion from peripheral blood mononuclear cells (PBMCs) of five normal volunteers. The concentration of Ig secreted in the medium after incubation of 15 days was found increased in a dose-dependent manner in the presence of Grp94, used at the final concentrations of 10 and 100 ng/ml. However, by measuring the Ig secretion at different incubation times, it was apparent that maximal percent stimulation by Grp94 occurred at 7 days, decreasing thereafter. In addition, the pattern of Ig secretion in time significantly differed in the presence of Grp94 with respect to that of control PBMCs. Grp94 also stimulated in a dose-dependent manner the PBMC proliferation, an effect that preceded the Ig secretion and was accompanied by morphological changes of cells similar to those induced by the pokeweed mitogen. Effects of Grp94 on PBMCs were mediated by an intense activation of the MEK-ERK1/2 pathway and by an increased expression of HSP90. Results indicate that Grp94 can activate the humoral response by a cytokine-like, cell-mediated mechanism that leads to an accelerated process of B cell maturation and differentiation.

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.

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.

Characterization of immune complexes of idiotypic catalytic and anti-idiotypic inhibitory antibodies in plasma of type 1 diabetic subjects.

An increase in proteolytic activity is an early common feature of diabetes, and is associated with the development of vascular complications. We performed an extensive proteomic investigation on plasma of type 1 diabetic subjects to discover why some of them apparently lacked any measurable proteolytic activity. Activity was found enclosed in immune complexes in which Fab/(Fab)(2) displayed a serine-like catalytic activity. Disaggregation of complexes by means of Protein G affinity chromatography led to the separation of free subunits of Fab, showing a specific amidolytic activity, from Fab that displayed activity on casein and remained closely complexed with whole IgG. On both types of Fab the serine catalytic site appeared to be the same, being located in close vicinity to the antigen-binding site. The distinct substrate specificity was due to the different conformation adopted by the catalytic site depending on the structure of Fab/(Fab)(2), whether in complexes or as free subunits. Catalytic Fab/(Fab)(2) originated from idiotypic antibodies developed against Grp94, identified as the primary antigen covalently complexed with Fab. Whole IgG present in immune complexes were instead mostly formed with anti-idiotypic antibodies developed against the adduct of Fab/(Fab)(2) with Grp94, and were responsible for blocking any catalytic activity. In dot-blot experiments with native Grp94, we confirmed that in any diabetic plasma circulated anti-Grp94, idiotypic, and anti-idiotypic antibodies.

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.

A heat shock protein70 fusion protein with alpha1-antitrypsin in plasma of type 1 diabetic subjects.

The recent observation that heat shock proteins (HSPs), mostly glucose regulated protein94 (Grp94) and HSP70, are present in plasma of Type 1 diabetic subjects as complexes with immunoglobulins, prompted us to investigate the nature and extent of this association, whether it represents HSP-induced activation of the immune system. Two complementary affinity chromatography procedures followed by immunoprecipitation and immunoblot analyses of HSP-enriched, plasma-purified peaks, revealed that HSPs were inextricably linked with IgG in SDS-resistant complexes from which proteins dissociate partially under reducing treatment. HSP70 was found also closely linked with alpha1-antitrypsin (alpha1AT) in a single protein having the mass of alpha1AT but elution characteristics different from those of normal alpha1AT. Immunoprecipitation with anti-HSP70 antibodies led to co-immunoprecipitation of the alpha1AT species linked to HSP70, thus confirming fusion of the proteins. The additional finding of circulating antibodies against the HSP70-alpha1AT protein supported its immunogenic properties with implications for diabetes and its complications.