Towards antigen-specific Tregs for type 1 diabetes: Construction and functional assessment of pancreatic endocrine marker, HPi2-based chimeric antigen receptor.

Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.

The Type 1 Diabetes-Resistance Locus Idd22 Controls Trafficking of Autoreactive CTLs into the Pancreatic Islets of NOD Mice.

Type 1 diabetes (T1D) has a strong genetic component. The insulin dependent diabetes (Idd)22 locus was identified in crosses of T1D-susceptible NOD mice with the strongly T1D-resistant ALR strain. The NODcALR-(D8Mit293-D8Mit137)/Mx (NOD-Idd22) recombinant congenic mouse strain was generated in which NOD mice carry the full Idd22 confidence interval. NOD-Idd22 mice exhibit almost complete protection from spontaneous T1D and a significant reduction in insulitis. Our goal was to unravel the mode of Idd22-based protection using in vivo and in vitro models. We determined that Idd22 did not impact immune cell diabetogenicity or ß cell resistance to cytotoxicity in vitro. However, NOD-Idd22 mice were highly protected against adoptive transfer of T1D. Transferred CTLs trafficked to the pancreatic lymph node and proliferated to the same extent in NOD and NOD-Idd22 mice, yet the accumulation of pathogenic CTLs in the islets was significantly reduced in NOD-Idd22 mice, correlating with disease resistance. Pancreatic endothelial cells from NOD-Idd22 animals expressed lower levels of adhesion molecules, even in response to inflammatory stimuli. Lower adhesion molecule expression resulted in weaker adherence of T cells to NOD-Idd22 endothelium compared with NOD-derived endothelium. Taken together, these results provide evidence that Idd22 regulates the ability of ß cell-autoreactive T cells to traffic into the pancreatic islets and may represent a new target for pharmaceutical intervention to potentially prevent T1D.

Self-Transducible Bimodal PDX1-FOXP3 Protein Lifts Insulin Secretion and Curbs Autoimmunity, Boosting Tregs in Type 1 Diabetic Mice.

Type 1 diabetes (T1D) is characterized by massive destruction of insulin-producing ß cells by autoreactive T lymphocytes, arising via defective immune tolerance. Therefore, effective anti-T1D therapeutics should combine autoimmunity-preventing and insulin production-restoring properties. We constructed a cell-permeable PDX1-FOXP3-TAT fusion protein (FP) composed of two transcription factors: forkhead box P3 (FOXP3), the master regulator of differentiation and functioning of self-tolerance-promoting Tregs, and pancreatic duodenal homeobox-1 (PDX1), the crucial factor supporting ß cell development and maintenance. The FP was tested in vitro and in a non-obese diabetic mouse T1D model. In vitro, FP converted naive CD4+ T cells into a functional "Treg-like" subset, which suppressed cytokine secretion, downregulated antigen-specific responses, and curbed viability of diabetogenic effector cells. In hepatic stem-like cells, FP potentiated endocrine transdifferentiation, inducing expression of Insulin2 and other ß lineage-specific genes. In vivo, FP administration to chronically diabetic mice triggered (1) a significant elevation of insulin and C-peptide levels, (2) the formation of insulin-containing cell clusters in livers, and (3) a systemic anti-inflammatory shift (higher Foxp3+CD4+CD25+ T cell frequencies, elevated rates of IL-10-producing cells, and reduced rates of IFN-γ-secreting cells). Overall, in accordance with its design, PDX1-FOXP3-TAT FP delivered both Treg-stabilizing anti-autoimmune and de novo insulin-producing effects, proving its anti-T1D therapeutic potential.

New Insights into Immunotherapy Strategies for Treating Autoimmune Diabetes.

Type 1 diabetes mellitus (T1D) is an autoimmune illness that affects millions of patients worldwide. The main characteristic of this disease is the destruction of pancreatic insulin-producing beta cells that occurs due to the aberrant activation of different immune effector cells. Currently, T1D is treated by lifelong administration of novel versions of insulin that have been developed recently; however, new approaches that could address the underlying mechanisms responsible for beta cell destruction have been extensively investigated. The strategies based on immunotherapies have recently been incorporated into a panel of existing treatments for T1D, in order to block T-cell responses against beta cell antigens that are very common during the onset and development of T1D. However, a complete preservation of beta cell mass as well as insulin independency is still elusive. As a result, there is no existing T1D targeted immunotherapy able to replace standard insulin administration. Presently, a number of novel therapy strategies are pursuing the goals of beta cell protection and normoglycemia. In the present review we explore the current state of immunotherapy in T1D by highlighting the most important studies in this field, and envision novel strategies that could be used to treat T1D in the future.

Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes.

Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and ß cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.

Dynamic interdomain interactions contribute to the inhibition of matrix metalloproteinases by tissue inhibitors of metalloproteinases.

Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.

Biochemical characterization of the cellular glycosylphosphatidylinositol-linked membrane type-6 matrix metalloproteinase.

Ubiquitously expressed membrane type-1 matrix metalloproteinase (MT1-MMP), an archetype member of the MMP family, binds tissue inhibitor of metalloproteinases-2 (TIMP-2), activates matrix metalloproteinase-2 (MMP-2), and stimulates cell migration in various cell types. In contrast with MT1-MMP, the structurally similar MT6-MMP associates with the lipid raft compartment of the plasma membrane using a GPI anchor. As a result, MT6-MMP is functionally distinct from MT1-MMP. MT6-MMP is insufficiently characterized as yet. In addition, a number of its biochemical features are both conflicting and controversial. To reassess the biochemical features of MT6-MMP, we have expressed the MT6-MMP construct tagged with a FLAG tag in breast carcinoma MCF-7 and fibrosarcoma HT1080 cells. We then used phosphatidylinositol-specific phospholipase C to release MT6-MMP from the cell surface and characterized the solubilized MT6-MMP fractions. We now are confident that cellular MT6-MMP partially exists in its complex with TIMP-2. Both TIMP-1 and TIMP-2 are capable of inhibiting the proteolytic activity of MT6-MMP. MT6-MMP does not stimulate cell migration. MT6-MMP, however, generates a significant level of gelatinolysis of the fluorescein isothiocyanate-labeled gelatin and exhibits an intrinsic, albeit low, ability to activate MMP-2. As a result, it is exceedingly difficult to record the activation of MMP-2 by cellular MT6-MMP. Because of its lipid raft localization, cellular MT6-MMP is inefficiently internalized. MT6-MMP is predominantly localized in the cell-to-cell junctions. Because MT6-MMP has been suggested to play a role in disease, including cancer and autoimmune multiple sclerosis, the identity of its physiologically relevant cleavage targets remains to be determined.

Internal cleavages of the autoinhibitory prodomain are required for membrane type 1 matrix metalloproteinase activation, although furin cleavage alone generates inactive proteinase.

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.

The Wnt/planar cell polarity protein-tyrosine kinase-7 (PTK7) is a highly efficient proteolytic target of membrane type-1 matrix metalloproteinase: implications in cancer and embryogenesis.

PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.

Isolation and characterization of selective and potent human Fab inhibitors directed to the active-site region of the two-component NS2B-NS3 proteinase of West Nile virus.

There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.

Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

Inflammatory proprotein convertase-matrix metalloproteinase proteolytic pathway in antigen-presenting cells as a step to autoimmune multiple sclerosis.

Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS.

Biochemical evidence of the interactions of membrane type-1 matrix metalloproteinase (MT1-MMP) with adenine nucleotide translocator (ANT): potential implications linking proteolysis with energy metabolism in cancer cells.

Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia.

OBJECTIVE: Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium. APPROACH AND RESULTS: A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0µm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330µm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622µm2) and lipids (30754 vs. 77317µm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls. CONCLUSIONS: Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

Transgenic Overexpression of Tissue-Nonspecific Alkaline Phosphatase (TNAP) in Vascular Endothelium Results in Generalized Arterial Calcification.

BACKGROUND: Ectopic vascular calcification is a common condition associated with aging, atherosclerosis, diabetes, and/or chronic kidney disease. Smooth muscle cells are the best characterized source of osteogenic progenitors in the vasculature; however, recent studies suggest that cells of endothelial origin can also promote calcification. To test this, we sought to increase the osteogenic potential of endothelial cells by overexpressing tissue-nonspecific alkaline phosphatase (TNAP), a key enzyme that regulates biomineralization, and to determine the pathophysiological effect of endothelial TNAP on vascular calcification and cardiovascular function. METHODS AND RESULTS: We demonstrated previously that mice transgenic for ALPL (gene encoding human TNAP) develop severe arterial medial calcification and reduced viability when TNAP is overexpressed in smooth muscle cells. In this study, we expressed the ALPL transgene in endothelial cells following endothelial-specific Tie2-Cre recombination. Mice with endothelial TNAP overexpression survived well into adulthood and displayed generalized arterial calcification. Genes associated with osteochondrogenesis (Runx2, Bglap, Spp1, Opg, and Col2a1) were upregulated in the aortas of endothelial TNAP animals compared with controls. Lesions in coronary arteries of endothelial TNAP mice showed immunoreactivity to Runx2, osteocalcin, osteopontin, and collagen II as well as increased deposition of sialoproteins revealed by lectin staining. By 23 weeks of age, endothelial TNAP mice developed elevated blood pressure and compensatory left ventricular hypertrophy with preserved ejection fraction. CONCLUSIONS: This study presented a novel genetic model demonstrating the osteogenic potential of TNAP-positive endothelial cells in promoting pathophysiological vascular calcification.

Autoimmune diabetes is suppressed by treatment with recombinant human tissue Kallikrein-1.

The kallikrein-kinin system (KKS) comprises a cascade of proteolytic enzymes and biogenic peptides that regulate several physiological processes. Over-expression of tissue kallikrein-1 and modulation of the KKS shows beneficial effects on insulin sensitivity and other parameters relevant to type 2 diabetes mellitus. However, much less is known about the role of kallikreins, in particular tissue kallikrein-1, in type 1 diabetes mellitus (T1D). We report that chronic administration of recombinant human tissue kallikrein-1 protein (DM199) to non-obese diabetic mice delayed the onset of T1D, attenuated the degree of insulitis, and improved pancreatic beta cell mass in a dose- and treatment frequency-dependent manner. Suppression of the autoimmune reaction against pancreatic beta cells was evidenced by a reduction in the relative numbers of infiltrating cytotoxic lymphocytes and an increase in the relative numbers of regulatory T cells in the pancreas and pancreatic lymph nodes. These effects may be due in part to a DM199 treatment-dependent increase in active TGF-beta1. Treatment with DM199 also resulted in elevated C-peptide levels, elevated glucagon like peptide-1 levels and a reduction in dipeptidyl peptidase-4 activity. Overall, the data suggest that DM199 may have a beneficial effect on T1D by attenuating the autoimmune reaction and improving beta cell health.

Preclinical characterization of recombinant human tissue kallikrein-1 as a novel treatment for type 2 diabetes mellitus.

Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D.

Nardilysin-dependent proteolysis of cell-associated VTCN1 (B7-H4) marks type 1 diabetes development.

T-cell responses directed against insulin-secreting pancreatic ß-cells are the key events highlighting type 1 diabetes (T1D). Therefore, a defective control of T-cell activation is thought to underlie T1D development. Recent studies implicated a B7-like negative costimulatory protein, V-set domain-containing T-cell activation inhibitor-1 (VTCN1), as a molecule capable of inhibiting T-cell activation and, potentially, an important constituent in experimental models of T1D. Here, we unravel a general deficiency within the VTCN1 pathway that is shared between diabetes-prone mice and a subset of T1D patients. Gradual loss of membrane-tethered VTCN1 from antigen-presenting cells combined with an increased release of soluble VTCN1 (sVTCN1) occurs in parallel to natural T1D development, potentiating hyperproliferation of diabetogenic T cells. Mechanistically, we demonstrate that the loss of membrane-tethered VTCN1 is linked to proteolytic cleavage mediated by the metalloproteinase nardilysin. The cleaved sVTCN1 fragment was detected at high levels in the peripheral blood of 53% T1D patients compared with only 9% of the healthy subjects. Elevated blood sVTCN1 levels appeared early in the disease progression and correlated with the aggressive pace of disease, highlighting the potential use of sVTCN1 as a new T1D biomarker, and identifying nardilysin as a potential therapeutic target.

Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B-NS3 proteinase.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B-NS3pro structure, we hypothesized that the G(22) residue and the negatively charged patch D(32)DD(34) of NS2B were part of an important configuration required for NS2B-NS3pro activity. Our experimental data confirmed that G(22) and D(32)DD(34) substitution for S and AAA, respectively, inactivated NS2B-NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B-NS3pro. Because of the significant level of homology in flaviviral NS2B-NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.