Insulin-degrading enzyme (IDE) as a modulator of microglial phenotypes in the context of Alzheimer's disease and brain aging.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved zinc-dependent metallopeptidase highly expressed in the brain, where its specific functions remain poorly understood. Besides insulin, IDE is able to cleave many substrates in vitro, including amyloid beta peptides, making this enzyme a candidate pathophysiological link between Alzheimer's disease (AD) and type 2 diabetes (T2D). These antecedents led us to address the impact of IDE absence in hippocampus and olfactory bulb. A specific induction of microgliosis was found in the hippocampus of IDE knockout (IDE-KO) mice, without any effects in neither hippocampal volume nor astrogliosis. Performance on hippocampal-dependent memory tests is influenced by IDE gene dose in 12-month-old mice. Furthermore, a comprehensive characterization of the impact of IDE haploinsufficiency and total deletion in metabolic, behavioral, and molecular parameters in the olfactory bulb, a site of high insulin receptor levels, reveals an unambiguous barcode for IDE-KO mice at that age. Using wildtype and IDE-KO primary microglial cultures, we performed a functional analysis at the cellular level. IDE absence alters microglial responses to environmental signals, resulting in impaired modulation of phenotypic states, with only transitory effects on amyloid-ß management. Collectively, our results reveal previously unknown physiological functions for IDE in microglia that, due to cell-compartment topological reasons, cannot be explained by its enzymatic activity, but instead modulate their multidimensional response to various damaging conditions relevant to aging and AD conditions.

Association between polymorphism rs2421943 of the insulin-degrading enzyme and schizophrenia: Preliminary report.

BACKGROUND: Insulin-degrading enzyme (IDE) is an important gene in studies of the pathophysiology of type 2 diabetes mellitus (T2DM). Recent studies have suggested a possible link between type 2 diabetes mellitus (T2DM) and the pathophysiology of schizophrenia (SZ). At the same time, significant changes in insulin-degrading enzyme (IDE) gene expression have been found in the brains of people with schizophrenia. These findings highlight the need to further investigate the role of IDE in schizophrenia pathogenesis. METHODS: We enrolled 733 participants from the Czech Republic, including 383 patients with schizophrenia and 350 healthy controls. Our study focused on the single nucleotide polymorphism (SNP) rs2421943 in the IDE gene, which has previously been associated with the pathogenesis of Alzheimer's disease. The SNP was analyzed using the PCR-RFLP method. RESULTS: The G allele of the rs2421943 polymorphism was found to significantly increase the risk of developing SZ (p < 0.01) when a gender-based analysis showed that both AG and GG genotypes were associated with a more than 1.55 times increased risk of SZ in females (p < 0.03) but not in males. Besides, we identified a potential binding site at the G allele locus for has-miR-7110-5p, providing a potential mechanism for the observed association. CONCLUSION: Our results confirm the role of the IDE gene in schizophrenia pathogenesis and suggest that future research should investigate the relationship between miRNA and estrogen influence on IDE expression in schizophrenia pathogenesis.

Cryptosporidium equi n. sp. (Apicomplexa: Cryptosporidiidae): Biological and genetic characterisations.

The horse genotype is one of three common Cryptosporidium spp. in equine animals and has been identified in some human cases. The species status of Cryptosporidium horse genotype remains unclear due to the lack of extensive morphological, biological, and genetic data. In the present study, we have conducted biological and whole genome sequence analyses of an isolate of the genotype from hedgehogs and proposed to name it Cryptosporidium equi n. sp. to reflect its common occurrence in equine animals. Oocysts of C. equi measured 5.12 ± 0.36 µm × 4.46 ± 0.21 µm with a shape index of 1.15 ± 0.08 (n = 50). Cryptosporidium equi was infectious to 3-week-old four-toed hedgehogs (Atelerix albiventris) and mice, with a prepatent period of 2-9 days and a patent period of 30-40 days in hedgehogs. It was not infectious to rats and rabbits. Phylogenetic analyses of small subunit rRNA, 70 kDa heat shock protein, actin, 60 kDa glycoprotein and 100 other orthologous genes revealed that C. equi is genetically distinct from other known Cryptosporidium species and genotypes. The sequence identity between C. equi and Cryptosporidium parvum genomes is 97.9%. Compared with C. parvum, C. equi has lost two MEDLE genes and one insulinase-like protease gene and gained one SKSR gene. In addition, 60 genes have highly divergent sequences (sequence differences ≥ 5.0%), including those encoding mucin-like glycoproteins, insulinase-like peptidases, and MEDLE and SKSR proteins. The genetic uniqueness of C. equi supports its increasing host range and the naming of it as a valid Cryptosporidium species. This is the first known use of whole genome sequence data in delineating new Cryptosporidium species.

Effects of Insulin-degrading enzyme on the proliferation of swine Sertoli cells.

Sertoli cells, the only somatic cells in testis seminiferous tubules, provide a supporting microenvironment for male germ cells and play essential roles in spermatogenesis. The insulin-degrading enzyme (IDE), a ubiquitous zinc peptidase of the inverzincin family, plays crucial role in sperm production, as IDE-knockout mice presented decreased testis weight and impaired sperm viability and morphology. However, whether and how IDE affects swine Sertoli cell proliferation remains unclear. Thus, in the present study, we aimed to evaluate the effects of IDE on the proliferation of swine Sertoli cells, as well as its underlying molecular mechanism. After knocking down IDE expression with small interfering RNA transfection, we analyzed the proliferation of swine Sertoli cells as well as the expression of related regulatory factors (WT1, ERK, and AKT). The results showed that IDE knockdown promoted swine Sertoli cell proliferation and increased WT1 expression, possibly through activating ERK and AKT. Overall, our findings suggest that IDE may be involved in male reproduction by regulating Sertoli cell proliferation, which provides new information to better understand the regulatory mechanisms of swine Sertoli cells and improve the reproductive traits of male pigs.

Long-Term High-Fat Diet Decreases Renal Insulin-Degrading Enzyme Expression and Function by Inhibiting the PPARγ Pathway.

SCOPE: Long-term high-fat diet (HFD) causes insulin resistance, which is a primary etiological factor in the development of obesity and type 2 diabetes mellitus. Impaired insulin clearance is not only a consequence but also a cause of insulin resistance. The kidney is a major site of insulin clearance, where the insulin-degrading enzyme (IDE) plays a vital role in the proximal tubule. Thus, the study investigates the role of renal IDE in the regulation of insulin resistance in HFD-induced obese mice. METHODS AND RESULTS: Twenty four-weeks of HFD in C57BL/6 mice causes insulin resistance and impaires insulin clearance, accompanied by a decrease in renal IDE expression and activity. Palmitic acid decreases IDE mRNA and protein expressions in HK-2 cells. RNA-Seq analysis found that the PPAR pathway is involved. 24-weeks of HFD decreases renal PPARγ, but not PPARα or PPARß/δ mRNA expression. The inhibition of IDE expression by palmitic acid is prevented by the PPARγ agonist rosiglitazone. The amount of PPARγ bound to the promoters of IDE is decreased in palmitic acid-treated cells. Rosiglitazone improves insulin clearance and insulin resistance and increases renal IDE expression in HFD fed-mice. CONCLUSION: Long-term HFD decreases renal IDE expression and activity, and causes insulin resistance, which involves PPARγ.

Induction of ICAM1 in Brain Vessels is Implicated in an Early AD Pathogenesis by Modulating Neprilysin.

Intercellular adhesion molecule 1 (ICAM1) is a vessel adhesion protein induced during brain vascular inflammation, which could be closely linked with the development of Alzheimer's disease (AD). This study investigated the effect of ICAM1 on amyloid-degrading enzymes (ADEs) in endothelial cells and their potential involvement in inflammation and AD progression. TNF-α treatment increased ICAM1 in human brain microvascular endothelial cells (HBMVECs) but decreased the neprilysin (NEP) protein level. Knock-down of ICAM1 using siRNA enhanced NEP, which increased the degradation of amyloid-ß. In the brains of 4-month-old AD transgenic mice (APPswe/PSEN1dE9), there were significantly higher levels of ICAM1 expression and amyloid deposits but lower levels of NEP and insulin-degrading enzymes (IDE), demonstrating an inverse correlation of ICAM1 with NEP and IDE expression. Further studies demonstrated significantly increased GFAP protein levels in the brain, specifically localized near blood vessels, of both TNF-α-injected and 4-month-old AD transgenic mice. Taken together, the induction of ICAM1 in endothelial cells suppresses NEP expression, accelerating the accumulation of amyloid-ß in blood vessels. It also enhances leukocyte adhesion to blood vessels stimulating the migration of leukocytes into the brain, subsequently triggering brain inflammation.

Sirt3 deficiency induced down regulation of insulin degrading enzyme in comorbid Alzheimer's disease with metabolic syndrome.

SIRT3 deacetylates mitochondrial proteins, thereby enhancing their function. We have previously demonstrated that Sirt3 gene deletion leads to brain mitochondrial dysfunction and neuroinflammation. We also reported that silencing of Sirt3 gene in APP/PS1 mice results in exacerbation of insulin resistance, neuroinflammation and ß amyloid plaque deposition. To further understand how metabolic syndrome and amyloid pathology interact, we performed RNA-seq analysis of the brain samples of APP/PS1/Sirt3-/- mice. Gene expression patterns were modulated in metabolic and inflammatory pathways by Sirt3 gene deletion, amyloid pathology, and the combination. Following Sirt3 gene deletion, a key finding was the decreased expression of insulin-degrading enzyme (IDE), an enzyme that regulates the levels of insulin and Aß peptides. Western diet feeding of Sirt3-/- and APP/PS1 mice resulted in decrease of IDE protein, parallel to Sirt3 downregulation. Conversely, activation of SIRT3 by nicotinamide riboside in vivo and in vitro resulted in IDE upregulation. SIRT3 activation in vivo also increased the levels of neprilysin, another Aß degrading enzyme and decreased the levels of BACE1 which generates Aß peptide suggesting SIRT3's role in amyloid plaque reduction. Our findings provide a plausible mechanism linking metabolic syndrome and amyloid pathology. SIRT3 may be a potential therapeutic target to treat AD.

Insulin-degrading enzyme ablation in mouse pancreatic alpha cells triggers cell proliferation, hyperplasia and glucagon secretion dysregulation.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by hyperglucagonaemia and perturbed function of pancreatic glucagon-secreting alpha cells but the molecular mechanisms contributing to these phenotypes are poorly understood. Insulin-degrading enzyme (IDE) is present within all islet cells, mostly in alpha cells, in both mice and humans. Furthermore, IDE can degrade glucagon as well as insulin, suggesting that IDE may play an important role in alpha cell function in vivo. METHODS: We have generated and characterised a novel mouse model with alpha cell-specific deletion of Ide, the A-IDE-KO mouse line. Glucose metabolism and glucagon secretion in vivo was characterised; isolated islets were tested for glucagon and insulin secretion; alpha cell mass, alpha cell proliferation and α-synuclein levels were determined in pancreas sections by immunostaining. RESULTS: Targeted deletion of Ide exclusively in alpha cells triggers hyperglucagonaemia and alpha cell hyperplasia, resulting in elevated constitutive glucagon secretion. The hyperglucagonaemia is attributable in part to dysregulation of glucagon secretion, specifically an impaired ability of IDE-deficient alpha cells to suppress glucagon release in the presence of high glucose or insulin. IDE deficiency also leads to α-synuclein aggregation in alpha cells, which may contribute to impaired glucagon secretion via cytoskeletal dysfunction. We showed further that IDE deficiency triggers impairments in cilia formation, inducing alpha cell hyperplasia and possibly also contributing to dysregulated glucagon secretion and hyperglucagonaemia. CONCLUSIONS/INTERPRETATION: We propose that loss of IDE function in alpha cells contributes to hyperglucagonaemia in type 2 diabetes.

Link of BIN1, CLU, and IDE Gene Polymorphisms with the Susceptibility of Alzheimer's Disease: Evidence from a Meta-analysis.

BACKGROUND: Alzheimer's disease (AD) is the most common form of neurodegenerative disorder. The association of BIN1, CLU, and IDE genetic polymorphisms with AD risk have been evaluated overtimes that produced conflicting outcomes. OBJECTIVE: We performed this meta-analysis to investigate the contribution of BIN1 (rs744373 and rs7561528), CLU (rs11136000 and rs9331888), and IDE (rs1887922) polymorphisms to AD risk. METHODS: From a systemic literature search up to July 15, 2021, we included 25 studies with rs744373, 16 studies with rs7561528, 37 studies with rs11136000, 16 studies with rs9331888, and 4 studies with rs1887922. To analyze the correlation, we constructed seven genetic models that used odds ratio and 95% confidence intervals. We used RevMan 5.4 for meta-analysis. RESULTS: Our study suggests that BIN1 rs744373 is associated with a significantly increased risk of AD in five genetic models (OR>1). Again, CLU rs11136000 showed reduced association in all genetic models (OR<1). CLU rs9331888 revealed an increased association in two models (OR>1). The IDE rs1887922 showed significantly increased risk in four models (OR>1). From subgroup analysis, a significantly increased risk of AD was observed in Caucasians and Asians for BIN1 rs744373. Again, BIN1 rs7561528 showed a significantly enhanced risk of AD only in Caucasians. CLU rs11136000 showed significantly reduced risk in Caucasians but rs9331888 showed increased risk in the same ethnicity. CONCLUSION: Our meta-analysis confirms the association of BIN1 rs744373, CLU rs9331888, and IDE rs1887922 polymorphisms with an increased risk of AD, especially in Caucasians. Again, CLU rs11136000 is associated with reduced AD risk in the overall population and Caucasians.

Possible Role of Insulin-Degrading Enzyme in the Physiopathology of Retinitis Pigmentosa.

Insulin-degrading enzyme (IDE) was named after its role as a proteolytic enzyme of insulin. However, recent findings suggest that IDE is a widely expressed, multitask protein, with both proteolytic and non-proteolytic functions. Here, we characterize the expression of IDE in the mammalian retina in both physiological and pathological conditions. We found that IDE was enriched in cone inner segments. IDE levels were downregulated in the dystrophic retina of several mouse models of retinitis pigmentosa carrying distinct mutations. In rd10 mice, a commonly studied mouse model of retinitis pigmentosa, treatment with an IDE activator (a synthetic peptide analog of preimplantation factor) delayed loss of visual function and preserved photoreceptor cells. Together, these results point to potential novel roles for IDE in retinal physiology and disease, further extending the list of diverse functions attributed to this enzyme.

Insulin-Degrading Enzyme, an Under-Estimated Potential Target to Treat Cancer?

Insulin-degrading enzyme (IDE) is a multifunctional protease due to the variety of its substrates, its various cellular locations, its conservation between species and its many non-proteolytic functions. Numerous studies have successfully demonstrated its implication in two main therapeutic areas: metabolic and neuronal diseases. In recent years, several reports have underlined the overexpression of this enzyme in different cancers. Still, the exact role of IDE in the physiopathology of cancer remains to be elucidated. Known as the main enzyme responsible for the degradation of insulin, an essential growth factor for healthy cells and cancer cells, IDE has also been shown to behave like a chaperone and interact with the proteasome. The pharmacological modulation of IDE (siRNA, chemical compounds, etc.) has demonstrated interesting results in cancer models. All these results point towards IDE as a potential target in cancer. In this review, we will discuss evidence of links between IDE and cancer development or resistance, IDE's functions, catalytic or non-catalytic, in the context of cell proliferation, cancer development and the impact of the pharmacomodulation of IDE via cancer therapeutics.

Polymorphism Rs2421943 of the Insulin-Degrading Enzyme Gene and the Risk of Late-Onset Alzheimer's Disease.

BACKGROUND: Insulin-degrading enzyme (IDE) is a widely distributed Zn2+-binding metalloprotease that cleaves multiple short and medium-sized peptides prone to form ß-structures. These include insulin and amyloid-ß peptides. Accumulation and fibrillation of amyloid-ß peptides leading to the formation of amyloid plaques is a characteristic sign of Alzheimer's disease (AD) pathology. OBJECTIVE: The study investigated the rs2421943 single nucleotide polymorphism (SNP) of the IDE gene as a risk factor for MCI (mild cognitive impairment) and AD. METHODS: Two independent groups of 1670 patients and controls were included. The AD group consisted of 595 patients and 400 controls; the MCI group involved 135 patients and 540 matched controls. PCR and restriction fragment length analysis were used to analyze the rs2421943 polymorphism. Using the miRBase and RNA22 prediction tools in silico indicated that the rs2421943 polymorphism is a potential target for a specific miRNA (hsa-miR-7110-5p). RESULTS: AG and GG genotypes of rs2421943 significantly increased the risk of AD, and the AG genotype increased the risk of MCI. It seems the G allele both increases the risk of AD and accelerates the transition through the MCI phase. In silico study revealed that rs2421943 is inside the sequence binding miRNA hsa-miR-7110-5p. The polymorphism could affect the rate of IDE pre-RNA (heterogeneous nuclear RNA, hnRNA) processing, resulting in slower translation, lower levels of IDE, deficient removal of amyloid-ß fragments, and greater risk of and/or accelerated progression of AD. CONCLUSION: GG and AG genotypes of the single nucleotide polymorphism rs2421943 of insulindegrading enzyme gene increase the risk of AD and MCI.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Insulin-Degrading Enzyme: Paradoxes and Possibilities.

More than seven decades have passed since the discovery of a proteolytic activity within crude tissue extracts that would become known as insulin-degrading enzyme (IDE). Certainly much has been learned about this atypical zinc-metallopeptidase; at the same time, however, many quite fundamental gaps in our understanding remain. Herein, I outline what I consider to be among the most critical unresolved questions within the field, many presenting as intriguing paradoxes. For instance, where does IDE, a predominantly cytosolic protein with no signal peptide or clearly identified secretion mechanism, interact with insulin and other extracellular substrates? Where precisely is IDE localized within the cell, and what are its functional roles in these compartments? How does IDE, a bowl-shaped protein that completely encapsulates its substrates, manage to avoid getting "clogged" and thus rendered inactive virtually immediately? Although these paradoxes are by definition unresolved, I offer herein my personal insights and informed speculations based on two decades working on the biology and pharmacology of IDE and suggest specific experimental strategies for addressing these conundrums. I also offer what I believe to be especially fruitful avenues for investigation made possible by the development of new technologies and IDE-specific reagents. It is my hope that these thoughts will contribute to continued progress elucidating the physiology and pathophysiology of this important peptidase.

Effects of Fasting and Feeding on Transcriptional and Posttranscriptional Regulation of Insulin-Degrading Enzyme in Mice.

Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed Zn2+-metallopeptidase that regulates hepatic insulin sensitivity, albeit its regulation in response to the fasting-to-postprandial transition is poorly understood. In this work, we studied the regulation of IDE mRNA and protein levels as well as its proteolytic activity in the liver, skeletal muscle, and kidneys under fasting (18 h) and refeeding (30 min and 3 h) conditions, in mice fed a standard (SD) or high-fat (HFD) diets. In the liver of mice fed an HFD, fasting reduced IDE protein levels (~30%); whereas refeeding increased its activity (~45%) in both mice fed an SD and HFD. Likewise, IDE protein levels were reduced in the skeletal muscle (~30%) of mice fed an HFD during the fasting state. Circulating lactate concentrations directly correlated with hepatic IDE activity and protein levels. Of note, L-lactate in liver lysates augmented IDE activity in a dose-dependent manner. Additionally, IDE protein levels in liver and muscle tissues, but not its activity, inversely correlated (R2 = 0.3734 and 0.2951, respectively; p < 0.01) with a surrogate marker of insulin resistance (HOMA index). Finally, a multivariate analysis suggests that circulating insulin, glucose, non-esterified fatty acids, and lactate levels might be important in regulating IDE in liver and muscle tissues. Our results highlight that the nutritional regulation of IDE in liver and skeletal muscle is more complex than previously expected in mice, and that fasting/refeeding does not strongly influence the regulation of renal IDE.

Effect of Global Brain Ischemia on Amyloid Precursor Protein Metabolism and Expression of Amyloid-Degrading Enzymes in Rat Cortex: Role in Pathogenesis of Alzheimer's Disease.

The incidence of Alzheimer's disease (AD) increases significantly following chronic stress and brain ischemia which, over the years, cause accumulation of toxic amyloid species and brain damage. The effects of global 15-min ischemia and 120-min reperfusion on the levels of expression of the amyloid precursor protein (APP) and its processing were investigated in the brain cortex (Cx) of male Wistar rats. Additionally, the levels of expression of the amyloid-degrading enzymes neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1), and insulin-degrading enzyme (IDE), as well as of some markers of oxidative damage were assessed. It was shown that the APP mRNA and protein levels in the rat Cx were significantly increased after the ischemic insult. Protein levels of the soluble APP fragments, especially of sAPPß produced by ß-secretase, (BACE-1) and the levels of BACE-1 mRNA and protein expression itself were also increased after ischemia. The protein levels of APP and BACE-1 in the Cx returned to the control values after 120-min reperfusion. The levels of NEP and ECE-1 mRNA also decreased after ischemia, which correlated with the decreased protein levels of these enzymes. However, we have not observed any changes in the protein levels of insulin-degrading enzyme. Contents of the markers of oxidative damage (di-tyrosine and lysine conjugates with lipid peroxidation products) were also increased after ischemia. The obtained data suggest that ischemia shifts APP processing towards the amyloidogenic ß-secretase pathway and accumulation of the neurotoxic Aß peptide as well as triggers oxidative stress in the cells. These results are discussed in the context of the role of stress and ischemia in initiation and progression of AD.

Degradation of Alzheimer's Amyloid-ß by a Catalytically Inactive Insulin-Degrading Enzyme.

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-ß (Aß). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aß degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aß(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aß(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aß fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aß(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aß(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aß degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.

From virus to diabetes therapy: Characterization of a specific insulin-degrading enzyme inhibitor for diabetes treatment.

Inhibition of insulin-degrading enzyme (IDE) is a possible target for treating diabetes. However, it has not yet evolved into a medical intervention, mainly because most developed inhibitors target the zinc in IDE's catalytic site, potentially causing toxicity to other essential metalloproteases. Since IDE is a cellular receptor for the varicella-zoster virus (VZV), we constructed a VZV-based inhibitor. We computationally characterized its interaction site with IDE showing that the peptide specifically binds inside IDE's central cavity, however, not in close proximity to the zinc ion. We confirmed the peptide's effective inhibition on IDE activity in vitro and showed its efficacy in ameliorating insulin-related defects in types 1 and 2 diabetes mouse models. In addition, we suggest that inhibition of IDE may ameliorate the pro-inflammatory profile of CD4+ T-cells toward insulin. Together, we propose a potential role of a designed VZV-derived peptide to serve as a selectively-targeted and as an efficient diabetes therapy.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Loss of postprandial insulin clearance control by Insulin-degrading enzyme drives dysmetabolism traits.

Systemic insulin availability is determined by a balance between beta-cell secretion capacity and insulin clearance (IC). Insulin-degrading enzyme (IDE) is involved in the intracellular mechanisms underlying IC. The liver is a major player in IC control yet the role of hepatic IDE in glucose and lipid homeostasis remains unexplored. We hypothesized that IDE governs postprandial IC and hepatic IDE dysfunction amplifies dysmetabolic responses and prediabetes traits such as hepatic steatosis. In a European/Portuguese population-based cohort, IDE SNPs were strongly associated with postprandial IC in normoglycemic men but to a considerably lesser extent in women or in subjects with prediabetes. Liver-specific knockout-mice (LS-IDE KO) under normal chow diet (NCD), showed reduced postprandial IC with glucose intolerance and under high fat diet (HFD) were more susceptible to hepatic steatosis than control mice. This suggests that regulation of IC by IDE contributes to liver metabolic resilience. In agreement, LS-IDE KO hepatocytes revealed reduction of Glut2 expression levels with consequent impairment of glucose uptake and upregulation of CD36, a major hepatic free fatty acid transporter. Together these findings provide strong evidence that dysfunctional IC due to abnormal IDE regulation directly impairs postprandial hepatic glucose disposal and increases susceptibility to dysmetabolic conditions in the setting of Western diet/lifestyle.