Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Changes in urine proteome accompanying diabetic nephropathy progression.

INTRODUCTION: Owing to the prevalence of type 2 diabetes, diabetic kidney disease (DKD) becomes the major cause of end-stage renal disease. The current markers of diabetic nephropathy are based on albuminuria and clinical signs of retinopathy. Sensitive and specific noninvasive diagnostic tools, unbiased by the presence of comorbidities, are needed, especially to detect the early stages of diabetic complications. OBJECTIVES: The aim of the study was to analyze changes in urinary protein excretion based on the stage of DKD using quantitative proteomics. PATIENTS AND METHODS: A total of 27 healthy controls were age- and sex-matched to 72 diabetes patients classified into 3 groups: no signs of retinopathy or nephropathy (n = 33), retinopathy but no microalbuminuria (n = 15), and diabetic nephropathy (DN) based on overt albuminuria or microalbuminuria with retinopathy (n = 24). To assess the intergroup differences, samples were partially pooled, tagged using 8-plex iTRAQ reagents, and the resulting peptide mixture was resolved by isoelectrofocusing. The obtained fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were analyzed using the MASCOT software and dedicated in-house proteomic data analysis programs. RESULTS: The changes in the urine proteome following DKD progression involved some known protein markers of DN and several other proteins. Decreased levels of some proteins are presumably related to impaired secretory function of other organs affected by diabetes. In particular, a diminished excretion of pancreatic amylase and deoxyribonuclease I suggested exocrine pancreatic insufficiency (EPI), coexisting with type 2 diabetes. CONCLUSIONS: A decrease in the urinary excretion of some pancreatic enzymes suggests EPI associated with diabetes. This hypothesis is yet to be verified; nevertheless, renal and extrarenal confounders must be considered when interpreting the results of quantitative urinary proteomics.  

Complement components, proteolysis­related, and cell communication­related proteins detected in urine proteomics are associated with IgA nephropathy.

INTRODUCTION: IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The first symptoms of IgAN are erytrocyturia or hematuria, proteinuria, and decline in renal function, or any combination of the above. One of the promising diagnostic methods is urine proteomics. OBJECTIVES: We studied urine proteomics in patients with IgAN and age- and sex­matched healthy controls. To minimize the risk of protein degradation, we proposed a new protocol for urine collection and preparation. PATIENTS AND METHODS: A total of 30 patients with IgAN and 30 controls were enrolled into the study. Thirty urine samples of the IgAN group were divided into 3 disease pooled samples (DPS I, II, and III) and 30 urine samples of the control group were divided into 3 control pooled samples (CPS I, II, and III). We used isoelectric focusing/liquid chromatography-mass spectrometry/mass spectrometry (IEF/LC­MS/MS) to detect all proteins larger than 10 kDa. RESULTS: Using qualitative analysis, we identified 761, 951, and 956 proteins in each of the 3 IEF/LC­MS/MS experiments. The results were combined, yielding a dataset with 1238 proteins identified by at least 2 peptides. The statistical analysis of the quantitative results revealed 18 proteins that were differently populated in the urine of IgAN patients compared with healthy controls. We found increased urinary concentrations of complement components, coagulation factors, extracellular matrix, intracellular, transmembrane, and other proteins in patients with IgAN. Some of them have never been linked to IgAN before. CONCLUSIONS: We demonstrated that urine proteomics is a promising tool for diagnosing and monitoring patients with IgAN.

Alzheimer's disease Abeta peptide fragment 10-30 forms a spectrum of metastable oligomers with marked preference for N to N and C to C monomer termini proximity.

Oligomers of Abeta peptide have been indicated recently as a possible main causative agent of Alzheimer's disease. However, information concerning their structural properties is very limited. Here Abeta oligomers are studied by non-covalent complexes mass spectrometry and disulfide rearrangement. As a model molecule, an Abeta fragment spanning residues 10-30 (Abeta10-30) has been used. This model peptide is known to contain the core region responsible for Abeta aggregation to fibrils. Non-covalent complexes mass spectrometry indicates that, at neutral pH, monomers are accompanied by oligomers up to hexamers of gradually decreasing population. H-2H exchange studies and direct monomer exchange rate measurements with the use of 15N labeled peptides and mass spectrometry show a fast exchange of monomeric units between oligomers. Disulfide exchange studies of cysteine tagged Abeta10-30 and its mutant show proximity of N-N and C-C termini of monomers in oligomers. The presented data underscore a dynamic character for pre-nucleation forms of Abeta, however, with a marked tendency for parallel strand orientation in oligomers.