OBJECTIVEApproximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ATP-driven pump that translocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized.RESEARCH DESIGN AND METHODSThe streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca2+ uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss.RESULTSAfter 6–7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca2+ was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO's effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction.CONCLUSIONSThese data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes.

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2Kd and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K d with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrindependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K d bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K d endocytosis. Upon increased expression of wild type APLP2, K d molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K d at the plasma membrane, facilitates uptake of K d in a clathrin-dependent manner, and routes the endocytosed K d to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.The efficacy of the cellular immune response to intracellular pathogens and tumors is reliant on major histocompatibility complex (MHC) 5 class I molecules. MHC class I molecules present peptides, including peptides derived from pathogens and tumors, at the cell surface to cytotoxic T lymphocytes.Cytotoxic T lymphocytes have been selected during development for their ability to react to cells in the periphery expressing self MHC class I molecules bearing non-self peptides. The level of cell surface expression of MHC class I molecules on infected and malignant cells therefore dictates the extent to which the antigen-specific cytotoxic T lymphocytes can recognize, and subsequently lyse, abnormal cells. The cell surface expression of MHC class I molecules is controlled by the quantity and quality of MHC class I molecules that are assembled, and also by the rate at which MHC class I molecules depart from the plasma membrane.Amyloid precursor-like protein 2 (APLP2) binds to the MHC class I molecule K d in cells that express ␤2-microglobulin (1, 2). APLP2 regulates the level of folded K d molecules at the cell surface: a reduction in APLP2 expression causes an elevation in the amount of folded K d at the plasma membrane, and an increase in APLP2 expression results in a decline in folded K d surface expression (3, 4). Higher APLP2 expression lowers the level of K d at the plasma membrane by increasing the endocytosis, destabilization, and turnover of K d molecules (4). The family of proteins to which APLP2 belongs includes APL-1 in Caenorhabditis elegans, APPL in Drosophila, and three proteins in mammals: amyloid precursor protein (APP), amyloid precursor-like protein 1, and APLP2 (5, 6). APLP2 shares a high degree of sequence homology with APP, particularly at the C-terminal end. However, ...

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.

The present study was undertaken to determine if carbonylation of SERCA2 contributes to the prolongation of active cardiac relaxation time during diabetes. Type 1 diabetes was induced in male Sprague‐Dawley rats using streptozotocin (STZ). After seven weeks, serum reactive carbonyl species (RCS) increased 300%. Cardiac transmitral valve flow pattern (E:A ratio) and myocyte relaxation rate were decreased by 25% and 22%. Myocyte evoked Ca2+‐transient decay time increased 3.8‐fold. Ca2+ uptake and SERCA2 Ca2+ATPase activity were reduced by 57% and 60%. SERCA2 expression decreased by 10% but phospholamban levels remained unchanged. Trypsin digestion followed by mass spectroscopic analysis revealed elevated levels of carbonyl adducts on select basic residues of SERCA2. Crosslinking pentosidine adducts were also found on SERCA2 using adduct‐specific antibodies. Incubating hSERCA2 with the methylglyoxal reduced its ability to transport Ca2+. Arg164(Y)E, Arg636(Y)E, Lys475(Y)E and Lys481(Y)E mutations to mimic adducts formed during diabetes reduced the ability of SERCA2 to transport Ca2+. Treating STZ‐diabetic rats with pyridoxamine for five weeks, starting two weeks after STZ injections reduced serum RCS levels and blunted SERCA2 activity loss without altering steady state levels of SERCA2 or phospholamban. Pyridoxamine treatment also attenuated carbonylation of SERCA2. These new data demonstrate that carbonylation of SERCA2 is functionally important and contributes to the prolongation of active cardiac relaxation time during diabetes (SUPPORTED BY ADA)