The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the ß-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability.

Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics.

Thioredoxins (TRXs) constitute attractive α/ß scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1-93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94-108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cß; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R(2) values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a K(D) value of 1.4 ± 1.0 µM and a second low-affinity (>150 µM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of ß-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit.

Protocol to study the oligomeric organization of single-span transmembrane peptides using molecular dynamics simulations.

Herein, you will find detailed information for the preparation of a coarse-grained array of peptides embedded in a lipid membrane. It contains all the steps to set up and run a molecular dynamic simulation using a coarse-grained approach. We provide analytical tools and scripts for generating a residue-level contact matrix between multiple peptides, as well as geometric analysis of arrangements between multiple peptides. This protocol was designed to study the organization of transmembrane peptides in an unbiased manner using computational approaches. For complete details on the use and execution of this protocol, please refer to Smulski et al. (2022).

Differential trafficking of ligands trogocytosed via CD28 versus CTLA4 promotes collective cellular control of co-stimulation.

Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is unclear whether they also jointly regulate their availability and degradation. Here we use newly developed reporter systems, bioinformatic analyses, protein structure modeling and genetic perturbations to assess this. We find that T cells utilize trogocytosis by competing antagonistic receptors to differentially control the abundance of immunoregulatory ligands. Specifically, ligands trogocytosed via CD28 are shuttled to the T cell surface, enabling them to co-stimulate neighboring T cells. In contrast, CTLA4-mediated trogocytosis targets ligands for degradation. Mechanistically, this fate separation is controlled by different acid-sensitivities of receptor-ligand interactions and by the receptor intracellular domains. The ability of CD28 and CTLA4 to confer different fates to trogocytosed ligands reveals an additional layer of collective regulation of cellular behaviors and promotes the robustness of population dynamics.

Ligand-independent oligomerization of TACI is controlled by the transmembrane domain and regulates proliferation of activated B cells.

In mature B cells, TACI controls class-switch recombination and differentiation into plasma cells during T cell-independent antibody responses. TACI binds the ligands BAFF and APRIL. Approximately 10% of patients with common variable immunodeficiency (CVID) carry TACI mutations, of which A181E and C172Y are in the transmembrane domain. Residues A181 and C172 are located on distinct sides of the transmembrane helix, which is predicted by molecular modeling to spontaneously assemble into trimers and dimers. In human B cells, these mutations impair ligand-dependent (C172Y) and -independent (A181E) TACI multimerization and signaling, as well as TACI-enhanced proliferation and/or IgA production. Genetic inactivation of TACI in primary human B cells impaired survival of CpG-activated cells in the absence of ligand. These results identify the transmembrane region of TACI as an active interface for TACI multimerization in signal transduction, in particular for ligand-independent signals. These functions are perturbed by CVID-associated mutations.

Coarse Grained Molecular Dynamic Simulations for the Study of TNF Receptor Family Members' Transmembrane Organization.

The Tumor Necrosis Factor (TNF) and the TNF receptor (TNFR) superfamilies are composed of 19 ligands and 30 receptors, respectively. The oligomeric properties of ligands, both membrane bound and soluble, has been studied most. However, less is known about the oligomeric properties of TNFRs. Earlier reports identified the extracellular, membrane-distal, cysteine-rich domain as a pre-ligand assembly domain which stabilizes receptor dimers and/or trimers in the absence of ligand. Nevertheless, recent reports based on structural nuclear magnetic resonance (NMR) highlight the intrinsic role of the transmembrane domains to form dimers (p75NTR), trimers (Fas), or dimers of trimers (DR5). Thus, understanding the structural basis of transmembrane oligomerization may shed light on the mechanism for signal transduction and the impact of disease-associated mutations in this region. To this end, here we used an in silico coarse grained molecular dynamics approach with Martini force field to study TNFR transmembrane homotypic interactions. We have first validated this approach studying the three TNFR described by NMR (p75NTR, Fas, and DR5). We have simulated membrane patches composed of 36 helices of the same receptor equidistantly distributed in order to get unbiassed information on spontaneous proteins assemblies. Good agreement was found in the specific residues involved in homotypic interactions and we were able to observe dimers, trimers, and higher-order oligomers corresponding to those reported in NMR experiments. We have, applied this approach to study the assembly of disease-related mutations being able to assess their impact on oligomerization stability. In conclusion, our results showed the usefulness of coarse grained simulations with Martini force field to study in an unbiased manner higher order transmembrane oligomerization.

Structural selection of a native fold by peptide recognition. Insights into the thioredoxin folding mechanism.

Thioredoxins (TRXs) are monomeric alpha/beta proteins with a fold characterized by a central twisted beta-sheet surrounded by alpha-helical elements. The interaction of the C-terminal alpha-helix 5 of TRX against the remainder of the protein involves a close packing of hydrophobic surfaces, offering the opportunity of studying a fine-tuned molecular recognition phenomenon with long-range consequences on the acquisition of tertiary structure. In this work, we focus on the significance of interactions involving residues L94, L99, E101, F102, L103 and L107 on the formation of the noncovalent complex between reduced TRX1-93 and TRX94-108. The conformational status of the system was assessed experimentally by circular dichroism, intrinsic fluorescence emission and enzymic activity; and theoretically by molecular dynamics simulations (MDS). Alterations in tertiary structure of the complexes, resulting as a consequence of site specific mutation, were also examined. To distinguish the effect of alanine scanning mutagenesis on secondary structure stability, the intrinsic helix-forming ability of the mutant peptides was monitored experimentally by far-UV CD spectroscopy upon the addition of 2,2,2-trifluoroethanol, and also theoretically by Monte Carlo conformational search and MDS. This evidence suggests a key role of residues L99, F102 and L103 on the stabilization of the secondary structure of alpha-helix 5, and on the acquisition of tertiary structure upon complex formation. We hypothesize that the transition between a partially folded and a native-like conformation of reduced TRX1-93 would fundamentally depend on the consolidation of a cooperative tertiary unit based on the interaction between alpha-helix 3 and alpha-helix 5.

Frequency of pancreatic beta-cell autoimmunity markers in patients with autoimmune thyroid disease.

A total of 305 ambulatory patients recruited at the Division of Endocrinology, Hospital de Clínicas, University of Buenos Aires, with autoimmune thyroid disease (AITD) were studied to search for associations between autoimmune thyroid disease and presence of serum markers of autoimmune diabetes mellitus. Screening for markers of pancreatic beta-cell autoimmunity was performed by radioligand binding assays (RBA) as follows: autoantibodies to glutamic acid decarboxylase (GADA) and proinsulin (PAA) were determined in all sera, whereas autoantibodies to protein tyrosine phosphatase (IA-2A) and insulin (IAA) were additionally measured in 200 sera randomly selected from the total collection. In addition, every GADA positive serum among the remaining 105 sera was systematically tested for the presence of IA-2A and IAA. In the cohort of 305 AITD patients 22 (7.2%) were previously diagnosed as type 1, type 2 or insulin-requiring type 2 diabetics. Ten of these patients presented serum marker positivity specific for beta-cell autoantigens and 12 were marker negative. On the other hand, considering the majority of non-diabetic AITD patients (n = 283), beta3-cell marker positivity was detected in 17 individuals (6.0%). The prevalence of autoimmune diabetes markers was much higher in the studied population than in the general population utilized as a control group, and GADA was the most frequent marker.

Development and characterisation of self-assembled graphene hydrogel-based anodes for bioelectrochemical systems.

In this work, we report a simple and scalable method to produce high efficiency 3D graphene-based electrodes (GH) for bioelectrochemical systems. GH were obtained by self-assembly of graphene oxide, through slow reduction with ascorbic acid over conductive mesh-works (carbon cloth and stainless-steel). The GH structure and composition were characterised by electron microscopy (SEM) and spectroscopy (FTIR and Raman), whereas the electrodes' performance was tested by chronoamperometry and cyclic voltammetry in a microbial electrolysis cell (MEC) inoculated with a pure culture of G. sulfurreducens. The hydrogel had a broad pore size distribution (>1 µm), which allowed bacterial colonisation within the framework. The macro-porous structure and chemical properties of the hydrogel rendered a higher bacterial loading capacity and substrate oxidation rate than other carbonaceous materials, including different reported graphene electrodes, which significantly increased MEC performance.

Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2.

The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic beta-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449-576 of IA-2 (IA-2ec(449-576)). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec(449-576) residues are alpha-helical, whereas about the same amount are in beta-sheet structure. The availability of soluble and folded IA-2ec(449-576) is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.

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.

Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

Structure of the mature ectodomain of the human receptor-type protein-tyrosine phosphatase IA-2.

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.

Effects of serine-to-cysteine mutations on beta-lactamase folding.

B. licheniformis exo-small beta-lactamase (ESBL) has two nonsequential domains and a complex architecture. We replaced ESBL serine residues 126 and 265 with cysteine to probe the conformation of buried regions in each domain. Spectroscopic, hydrodynamic, and chemical methods revealed that the mutations do not alter the native fold but distinctly change stability (S-126C > wild-type > S-126/265C > S-265C ESBL) and the features of partially folded states. The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure. S-126C ESBL intermediate has the fluorescence of the unfolded state, no thiol reactivity, and partial secondary structure. S-265C and S-126/265C ESBL populate intermediate states unfolded by fluorescence and thiol reactivity but with full secondary structure. Mass analysis of S-126/265C ESBL in the partially folded state proved that both thiol groups become exposed simultaneously. None of the intermediates is compatible with sequential domain unfolding. Molecular dynamics simulation suggests that the stabilizing effect of the S-126C substitution is due to optimization of van der Waals interactions and packing. On the other hand, destabilization induced by the S-265C mutation results from alteration of the hydrogen-bond network. The results illustrate the large impact that seemingly conservative serine-to-cysteine changes can have on the energy landscape of proteins.

High-yield expression of properly folded insulinoma-associated protein intracellular domain (IA-2ic) in Escherichia coli.

The intracellular domain of insulinoma-associated protein (IA-2), IA-2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease. The high-yield expression of properly folded IA-2ic is needed for basic research and crucial for low-cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine. In previous work, the expression of IA-2ic fused to glutathione S-transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield. Here we show that, utilizing a codon-optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained. Furthermore, the addition of a C-terminal His-tag greatly facilitates IA-2ic purification without compromising either its immunoreactivity or its expression yield. To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.

Frequency Of Pancreatic Beta-Cell Autoimmunity Markers In Patients With Autoimmune Thyroid Disease / Frecuencia de marcadores de autoinmunidad beta pancreática en pacientes con enfermedad tiroidea autoinmune

A total of 305 ambulatory patients recruited at the Division of Endocrinology, Hospital de Clínicas, University of Buenos Aires, with autoimmune thyroid disease (AITD) were studied to search for associations between autoimmune thyroid disease and presence of serum markers of autoimmune diabetes mellitus. Screening for markers of pancreatic beta-cell autoimmunity was performed by radioligand binding assays (RBA) as follows: autoantibodies to glutamic acid decarboxylase (GADA) and proinsulin (PAA) were determined in all sera, whereas autoantibodies to protein tyrosine phosphatase (IA-2A) and insulin (IAA) were additionally measured in 200 sera randomly selected from the total collection. In addition, every GADA positive serum among the remaining 105 sera was systematically tested for the presence of IA-2A and IAA. In the cohort of 305 AITD patients 22 (7.2%) were previously diagnosed as type 1, type 2 or insulin-requiring type 2 diabetics. Ten of these patients presented serum marker positivity specific for β-cell autoantigens and 12 were marker negative. On the other hand, considering the majority of non-diabetic AITD patients (n=283), β-cell marker positivity was detected in 17 individuals (6.0%). The prevalence of autoimmune diabetes markers was much higher in the studied population than in the general population utilized as a control group, and GADA was the most frequent marker.

Se investigó la asociación entre enfermedad tiroidea autoinmune y la presencia de marcadores séricos de diabetes mellitus en 305 pacientes ambulatorios con enfermedad tiroidea autoinmune reclutados en la División Endocrinología. La búsqueda de marcadores de autoinmunidad contra las células beta pancreáticas se realizó por la técnica de unión de radioligandos (RBA) como se detalla a continuación: se determinaron autoanticuerpos contra la decarboxilasa del ácido glutámico (GADA) y proinsulina (PAA) en todos los sueros, mientras que los anticuerpos contra la proteína tirosina fosfatasa (IA-2A) e insulina (IAA) fueron medidos en 200 de estos sueros tomados al azar de la colección total. Además, en los restantes 105 pacientes, la presencia de IA-2A y IAA fue evaluada en todos los sueros positivos para GADA. Del grupo de 305 pacientes con enfermedad tiroidea autoinmune 22 (7.2%) fueron diagnosticados previamente como diabéticos tipo 1, tipo 2 o tipo 2 insulino-requirientes. Diez de ellos presentaron positividad para marcadores específicos de autoantígenos de célula β, en tanto 12 fueron negativos. Por otra parte, en 17 de los 283 pacientes (6.0%) con enfermedad tiroidea autoimmune y sin diagnóstico previo de diabetes, se detectó positividad para marcadores de célula β. La prevalencia de marcadores de autoinmunidad asociados a diabetes fue mayor en la población estudiada que en la población general usada como grupo control, siendo GADA el marcador más frecuente.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (Type 1) in a argentine population

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, ainsulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1 por ciento for ICA, 36.7 percent for IAA, 74.6 por ciento for GADA and 63.4 por ciento ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8 por ciento, similar to the ICA512A- GADA (87.3 percent) or ICA512A-GADA-IAA combination (91.1 por ciento ). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4 percent) were positive for IAA and a single case (1.5 percent) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

Marcadores inmunológicos y genéticos en diabetes mellitus tipo 1 en Argentina / Immunologic and genetic markers in type 1 diabetes mellitus in Argentina

El objetivo del trabajo fue evaluar la prevalencia y asociación de los marcadores inmunológicos (anticuerpo anti-islote pancreático: ICA, autoanticuerpo anti-insulina: IAA, anticuerpo antidecarboxilasa del ácido glutámico: GADA y anticuerpo anti ICA512) y con el genotipo HLA DQBl en pacientes con diabetes tipo 1 de reciente debut, hermanos de diabéticos y personas sin historia de enfermedad autoinmune en población argentina. Se estudiaron 79 niños con diabetes tipo 1 de reciente debut, 79 niños controles y 68 hermanos sanos de niños con diabetes 1. En todos ellos se determinó IAA, GADA, ICA, ICA512 y alelos HLA DQB1. La sensibilidad para ICA fue de 67.1 por ciento; para IAA de 36,7 por ciento; para GADA de 74,6 por ciento, y para ICA512 de 63,4 por ciento. Ninguno de los niños control presentó marcadores inmunológicos positivos. La sensibilidad combinada de ICA-IAA-GADA fue de 89,8 por ciento, similar a la de ICA512-GADA (87.3 por ciento) o la combinación de ICA512-GADA-IAA (91.1 por ciento). El valor de GADA presentó correlación positiva con el de ICA, no encontrándose correlación alguna entre los valores de IAA, ICA512 e ICA. El valor de IAA presentó correlación negativa y el de GADA positiva con la edad de los pacientes. La presencia de IAA se asoció con DQB1 *0201, mientras que la de ICA e ICA512 con DQB1 *0302. Entre los hermanos, 3/68 (4,4 por ciento) fueron positivos para IAA, uno (1,5 por ciento) lo fue para GADA y otro (1.5 por ciento) para ICA512. Nuestros resultados muestran que la combinación de múltiples marcadores incrementa la sensibilidad predictiva, siendo la asociación ICA512-GADA altamente sensible y equivalente a otras combinaciones propuestas como ICA-IAA-GADA