ICA512 RESP18 homology domain is a protein-condensing factor and insulin fibrillation inhibitor.

Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase-like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet ß-cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named "regulated endocrine-specific protein 18 homology domain" (RESP18HD), which encompasses residues 35-131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91-131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other ß-cell proteins in a pH and Zn2+-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn2+ concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35-90, preceding the IDR, inhibit insulin fibrillation in vitro Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Condensation of the ß-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

cAMP-dependent phosphorylation of PTB1 promotes the expression of insulin secretory granule proteins in beta cells.

Glucose stimulates the exocytosis of insulin secretory granules of pancreatic beta cells. Granule stores are quickly refilled by activation of posttranscriptional mechanisms that enhance the biosynthesis of granule components. Rapid replacement of granules is important to sustain insulin secretion, since new granules appear to be preferentially released. Posttranscriptional regulation of granule biogenesis includes the glucose-induced nucleocytoplasmic translocation of polypyrimidine tract binding protein 1 (PTB1), which binds mRNAs encoding granule proteins, and thus promotes their stabilization and translation. Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin gene expression and secretion by increasing cAMP levels in beta cells. Here, we show that elevation of cAMP levels causes the protein kinase A-dependent phosphorylation and nucleocytoplasmic translocation of PTB1, thereby preventing the rapid degradation of insulin mRNA and enhancing the expression of various granule proteins. Taken together, these findings identify PTB1 as a common downstream target of glucose and GLP-1 for the posttranscriptional upregulation of granule biogenesis.

Stability of proICA512/IA-2 and its targeting to insulin secretory granules require ß4-sheet-mediated dimerization of its ectodomain in the endoplasmic reticulum.

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that ß2- or ß4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 ß2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 ß4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 ß2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 ß4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules.

PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells.

Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3'-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5'-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure.

Impaired insulin turnover in islets from type 2 diabetic patients.

Failure of pancreatic ß-cells contributes to the development of type 2 diabetes. Besides evidence of reduced glucose-stimulated insulin secretion and ß-cell mass, little information is available about the molecular deficits of human diabetic islets. Islets were isolated from macroscopically normal pancreatic tissue from 8 patients with type 2 diabetes and 17 matched non-diabetic patients who underwent pancreatic surgery. Insulin content and insulin secretion were measured before and after islet stimulation with 25 mM glucose for 2 hours. In parallel, we also investigated the subcellular localization of polypyrimidine tract-binding protein 1 (PTBP1), whose nucleocytoplasmic translocation is involved in the rapid posttranscriptional up-regulation of insulin biosynthesis following islet stimulation with glucose and GLP-1. Glucose stimulated insulin secretion was decreased, albeit not significantly, in type 2 diabetic islets compared to non-diabetic islets. Stimulation increased the total amount of insulin (islet insulin content + secreted insulin) in islet preparation from non-diabetic patients, but not from type 2 diabetic subjects. Furthermore, the nuclear levels of PTBP1 were decreased in stimulated non-diabetic islets, but not in type 2 diabetic islets. These results suggest that impairment of rapid insulin increase in response to glucose is a specific trait of type 2 diabetic islets. Nuclear retention of PTBP1 is likely to play a role in this deficit, which in turn can contribute to impaired insulin secretion in type 2 diabetes. Overall, these data highlight the importance of investigating mechanisms of insulin biosynthesis and degradation to gain insight into the pathogenesis of type 2 diabetes.

ß2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic ß-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of ß2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of ß2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-ß2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that ß2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and ß2-syntrophin phosphomutants we found that phosphorylation of ß2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.