Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation

BACKGROUND: Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM. METHODS: Experiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student’s t-tests or ANOVA and p-values of < 0.05 have been considered significant. RESULTS: Left-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks’ diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A). CONCLUSIONS: Myocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding

Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development

Diabetes-induced alterations in tissue collagen and carboxymethyllsine in rat kidneys: Association with increased collagent-degrading proteinases and amelioration by Cu(II)-slective chelation

AbstractAdvanced glycation end-products (AGEs) comprise a group of non-enzymatic post-translational modifications of proteins and are elevated in diabetic tissues. AGE-modification impairs the digestibility of collagen in vitro but little is known about its relation to collagen-degrading proteinases in vivo. Nε-carboxymethyllysine (CML) is a stable AGE that forms on lysyl side-chains in the presence of glucose, probably via a transition metal-catalysed mechanism.Here, rats with streptozotocin-induced diabetes and non-diabetic controls were treated for 8weeks with placebo or the Cu(II)-selective chelator, triethylenetetramine (TETA), commencing 8weeks after disease induction. Actions of diabetes and drug treatment were measured on collagen and collagen-degrading proteinases in kidney tissue.The digestibility and CML content of collagen, and corresponding levels of mRNAs and collagen, were related to changes in collagen-degrading-proteinases. Collagen-degrading proteinases, cathepsin L (CTSL) and matrix metalloproteinase-2 (MMP-2) were increased in diabetic rats. CTSL-levels correlated strongly and positively with increased collagen-CML levels and inversely with decreased collagen digestibility in diabetes. The collagen-rich mesangium displayed a strong increase of CTSL in diabetes. TETA treatment normalised kidney collagen content and partially normalised levels of CML and CTSL.These data provide evidence for an adaptive proteinase response in diabetic kidneys, affected by excessive collagen-CML formation and decreased collagen digestibility. The normalisation of collagen and partial normalisation of CML- and CTSL-levels by TETA treatment supports the involvement of Cu(II) in CML formation and altered collagen metabolism in diabetic kidneys. Cu(II)-chelation by TETA may represent a treatment option to rectify collagen metabolism in diabetes independent of alterations in blood glucose levels

Mass spectrometry-based determination of APLP2 γ-cleavage site.

<p>(<b>A</b>) Scheme of experimental procedure: the HA-APLP2CTF-FLAG construct was stably expressed in HEK293 cells. The construct consists of an N-terminal HA-tag (HA), a linker region consisting of five glycines (5G), the APLP2CTF sequence (APLP2) and an C-terminal FLAG-tag (FLAG). Upon γ-secretase cleavage, peptides harboring the γ-secretase cleavage site at their C-terminus were liberated. Peptides were immunoprecipitated using HA-affinity agarose and subsequently analyzed in a MALDI-TOF mass spectrometer. (<b>B</b>) Western Blot to confirm the γ-secretase sensitivity of the construct. Cells stably expressing HA-APLP2CTF-FLAG were treated with (M) or without (C, control) the γ-secretase inhibitor Merck A. In the presence of Merck A the CTFs accumulated in the lysate. The same was observed for cells grown in ‘light’ (L) or ‘heavy’ (H) medium. CTFs were detected with anti-FLAG antibody. Calnexin levels were analyzed as a loading control. (<b>C</b>+<b>D</b>) Mass spectrometry-based identification of the γ-secretase cleavage sites. Under control conditions, seven peaks were detected with a maximal total ion count of 497. Upon Merck A treatment, the intensity (ion count) for the peaks was reduced significantly. Peaks are labeled with identifiers #1–#7 linking them to the respective peptides sequences in the table (D). All peptides were detected with a mass error of less than 0.3 Da. (<b>E</b>) Determination of the labeling efficiency for the SILAC experiment. To determine the labeling efficiency, proteins from the cell lysates of the ‘heavy’ labeled cells from (F) were separated on an SDS-PAGE gel. One band was cut out and tryptic in gel digestion was performed. Peptides resulting from this digestion were analyzed in an LTQ Orbitrap Velos mass spectrometer. Proteins were identified by database search, and the labeling efficiency (Label. Eff.) was determined from the ‘heavy’ to ‘light’ ratios (Heavy/Light) of the quantifiable peptides. Further, coverage of the proteins in % (Coverage), number of unique peptides identified (Peptides), molecular weight of respective protein (MW), SEQUEST score (Score) and number of unique peptides used for quantification (H/L Count) are given. (<b>F</b>) Analysis of γ-secretase-dependent CTF cleavage by quantitative mass spectrometry. Upper panel: Cells without Merck A (Control) grown either in ‘light’ or in ‘heavy’ medium. Supernatants were combined, peptides immunoprecipitated and analyzed in the MALDI-TOF mass spectrometer. Peaks resulting from the ‘light’ and the ‘heavy’ labeled peptides are clearly separated (+10 Da as expected for one heavy labeled arginine). Intensities of corresponding ‘heavy’ and ‘light’ peak clusters are not identical, as the ‘heavy’ clusters are either shifted (+10 Da) or supershifted (+16 Da) due to additional incorporation of a ‘heavy’ proline resulting from arginine- to-proline conversion. Lower panel: upon treatment of the ‘light’ labeled cells with Merck A and the ‘heavy’ labeled cells with DMSO as a solvent control, the ratio between the intensity of the ‘light’ labeled peptides and the ‘heavy’ labeled peptides is significantly reduced if compared to the control experiment. This demonstrates that the identified peptide peaks are generated by γ-secretase. (<b>G</b>) Comparison of the position of the ε- and γ-secretase cleavage sites of APP and APLP2 in an alignment of the juxtamembrane and the transmembrane region of these two proteins. The dark boxes mark the predicted transmembrane domains (TMD), while the light box indicates amino acids which could potentially still be part of the transmembrane domain, as they do not harbor charges.</p

Analysis of APLP2 shedding using protease inhibitors.

<p>(<b>A</b>) Human neuroblastoma SH-SY5Y cells were transiently transfected with an siRNA pool against APLP2 or with a control pool. Endogenous APLP2 was detected with the N-terminally binding 2D11 antibody. Bands were absent upon knock-down of APLP2 (APLP2KD), demonstrating the specificity of the antibody. Soluble APLP2 (sAPLP2) was detected in the conditioned medium, cellular full-length APLP2 (cell. APLP2), cellular full-length APP (cell. APP) and calnexin as a loading control in the cell lysate. Three forms of APLP2 were detected: CS-GAG modified (*) and two non-CS-GAG modified species (** and ***) with molecular weights of around 115 and 100 kDa respectively. (<b>B</b>) Deglycosylation of APLP2 in conditioned medium and cell lysate of SH-SY5Y cells using endoglycosidase H (H) and N-glycosidase F (F). Deglycosylated forms of APLP2 are indicated (-- and ---). As a control, deglycosylation was performed for BACE1 in the cell lysate of BACE1 overexpressing HEK293 cells. Mature (#), immature (##) and deglycosylated (###) BACE1 is detectable. (<b>C</b>) Representative blots of treatment of SH-SY5Y cells with C3 (1 µM), TAPI-1 (50 µM) or C3+TAPI-1. Upon C3 treatment no significant reduction of sAPLP2 levels was observed. Upon TAPI-1 and C3+TAPI-1 treatment sAPLP2 levels were strongly reduced while no changes in APLP2 levels in the cell lysate were observed. As a control, soluble APP (sAPP) levels were clearly reduced upon TAPI-1 and C3+TAPI-1 treatment. (<b>D</b>) Quantification of experiments in C (mean +/− SEM). C3 treatment did not lead to a significant reduction in sAPLP2 levels while TAPI-1 as well as C3+TAPI-1 treatment led to a significant reduction in sAPLP2 levels (p<0.001 for all three species, n = 6).</p

Transient and stable knock-down of ADAM10 suppresses APLP2 shedding.

<p>(<b>A</b>) SH-SY5Y cells were transfected with siRNA pools against the proteases ADAM10 (A10KD) or ADAM17 (A17KD) or with control siRNA (Con). Both proteases were detected in membrane preparations. The mature active form is indicated with ##, the immature form with #. Actin and full-length APLP2 levels (cell. APLP2) were detected in the cell lysate. Conditioned media were analyzed for total secreted APLP2 (sAPLP2). All three species of sAPLP2 (* CS-GAG modified, ** 115 kDa, *** 100 kDa) were strongly decreased upon knock-down of ADAM10, but not of ADAM17. (<b>B</b>) Quantification of experiments in A (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for all three species, n = 6), while ADAM17 knock-down did not lead to any significant changes in sAPLP2 levels. (<b>C</b>) Knock-down of ADAM10 and ADAM17 in HEK293 cells, carried out as in A. (<b>D</b>) Quantification of experiments in C (mean +/− SEM). ADAM10 knock-down significantly reduced sAPLP2 (p<0.001 for CS-GAG modified and 100 kDa APLP2, p = 0.001 for 115 kDa APLP2, n = 6). (<b>E</b>) SH-SY5Y cells with stable shRNA-mediated knock-down of ADAM10 were used. shRNAs sh7 and sh9 are targeting two different regions of ADAM10. As control, a stable SH-SY5Y cell line expressing a non-targeting shRNA was used (Con). sAPLP2 levels were clearly reduced upon ADAM10 knock-down. (<b>F</b>) Quantification of experiments in E (mean +/− SEM). Both shRNAs significantly reduced sAPLP2 (p<0.001 for all three species, n = 6). (<b>G</b>) SH-SY5Y cells were transiently transfected with a siRNA pool against ADAM10 (A10KD) or control siRNA (Con). Cells were treated with DAPT (1 µM). Additionally C3 (1 µM) or DMSO (as a solvent control) was applied. sAPLP2 levels were clearly reduced upon ADAM10 knock-down while cellular APLP2 levels (cell. APLP2) remained unchanged. Two forms of APLP2 C-terminal fragments (CTFs) were detected at around 10 kDa in the cell lysate (-,--). ADAM10 knock-down led to the elimination of the lower molecular weight APLP2 CTF (--) while C3-treatment abolished the higher molecular weight species (-).</p

Mass spectrometry-based determination of APLP2 α- and β-shedding sites.

<p>(<b>A</b>) Scheme of experimental procedure. A TEV-protease cleavage site followed by a FLAG-tag was introduced into APLP2 isoform 763 starting after amino acid M653 (APLP2TF). The TEV-FLAG site is positioned 39 amino acids N-terminally of the assumed start of the transmembrane domain (TMD). Shedding yields sAPLP2TF, which was immunoprecipitated and digested with TEV-protease, leading to a small peptide harboring the N-terminal FLAG-tag as well as the C-terminal cleavage site resulting from the shedding. This peptide was analyzed in a mass spectrometer. (<b>B</b>) HEK293 cells were transiently transfected with the APLP2TF construct and either treated with TAPI-1 (50 µM) or DMSO as a solvent control. Upon TAPI-1 treatment secreted sAPLP2TF levels were clearly decreased in the conditioned medium of the cells. An accumulation of APLP2TF levels was observed upon TAPI-1 treatment in the cell lysate. Actin was analyzed in the cell lysate as a loading control. (<b>C</b>) Peptides produced according to the scheme in A were obtained from HEK293 cells transiently overexpressing APLP2TF and either luciferase as a control, ADAM10 or BACE1. Mass spectrometric analysis of the peptides revealed two peaks for control and ADAM10 overexpression (*,**) while upon BACE1 overexpression only one peak with a lower m/z ratio was observed. Total Ion Counts (TIC) and centroid peak masses of the first isotopic peak are given for each isotopic peak cluster. All peaks are revealed from singly-charged peptides. (<b>D</b>) Determined (Det.) masses of the peaks in C (*,**,***) were compared to calculated (calc.) masses. For each mass, a corresponding peptide could be computed with less than 0.5 Da error. (<b>E</b>) Western Blot analysis of the samples used in C. sAPLP2TF was detected in the conditioned media, full-length APLP2 (APLP2TF), ADAM10 (A10, # immature and ## mature), BACE1 and calnexin as a loading control were detected in the cell lysates. A clear increase in sAPLP2TF levels was observed upon ADAM10 and BACE1 overexpression. sAPLP2TF has a slightly reduced apparent molecular weight upon BACE1 overexpression if compared to control (Con). (<b>F</b>) Summary of known ADAM10 cleavage sites in different substrates aligned with the newly detected ADAM10 cleavage site for APLP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021337#pone.0021337-Caescu1" target="_blank">[41]</a>. (<b>G</b>) Schematic comparison of the ectodomain shedding sites of APP and APLP2. The APP α-cleavage occurs 12 amino acids N-terminally of the transmembrane domain (TMD), for APLP2 it occurs 22 amino acids N-terminally of the TMD. For both proteins β-cleavage occurs N-terminally of an aspartate.</p