Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes.

We purified to homogeneity and characterized NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. SDS/PAGE and immunoblotting with antibodies specific to these enzymes revealed a molecular mass estimated at 72 kDa for NTPDase1 and 66 for NTPDase2. Both enzymes exhibited kinetic properties typical for all members of the NTPDase family, e.g. low substrate specificity for tri- and diphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. The calculated Km value for NTPDase1 in respect to ATP as a substrate (97 microm) was three times lower in comparison to analogous values for NTPDase2 (270 microm). Additionally, NTPDase1 had a three times higher Kcat/Km coefficient than NTPDase2 (860 and 833 micromol product.s(-1), respectively). We have also demonstrated that in spite of differences in the affinity of ATP for both hydrolases, these enzymes have similar molecular activity. Taken together, these results indicate that NTPDase1 would terminate P2 receptor-mediated signal transmission whereas activity of NTPDase2 may contribute to decreasing high (toxic) concentrations of ATP and/or to production of another signal molecule, ADP.     

Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8

Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage.     

Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis (P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis (P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate (P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5-di(adenosine-5')pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 microg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K(m) values of 77.6+/-10.2 and 106.8+/-21.0 microM, and V(max) values of 68.9+/-8.1 and 99.4+/-8.5 (mean+/-S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72+/-0.24% (mean+/-S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.     

Conserved lysine 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

Cell membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are homooligomeric, with native quaternary structure required for maximal enzyme activity. In this study, we mutated lysine 79 in human ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). The residue corresponding to lysine 79 in NTPDase3 is conserved in all known cell surface membrane NTPDases (NTPDase1, 2, 3, and 8), but not in the soluble, monomeric NTPDases (NTPDase5 and 6), or in the intracellular, two transmembrane NTPDases (NTPDase4 and 7). This conserved lysine is located between apyrase conserved region 1 (ACR1) and an invariant glycosylation site (N81), in a region previously hypothesized to be important for NTPDase3 oligomeric structure. This lysine residue was mutated to several different amino acids, and all mutants displayed substantially decreased nucleotidase activities. A basic amino acid at this position was found to be important for the increase of nucleotidase activity observed after treatment with the lectin, concanavalin A. After solubilization with Triton X-100, mutants showed little or no decrease in activity, unlike the wild-type enzyme, suggesting that the lysine at this position may be important for maintaining proper folding and for stabilizing the quaternary structure. However, mutation at this site did not result in global changes in tertiary or quaternary structure as measured by Cibacron blue binding, chemical cross linking, and native gel electrophoretic analysis, leaving open the possibility of other mechanisms by which mutation of this conserved lysine residue might decrease enzyme activity.     

Localization of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and NTPDase2 in pancreas and salivary gland.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are membrane-bound ectoenzymes that hydrolyze extracellular nucleotides. We investigated the distribution of NTPDase1 and NTPDase2 in murine salivary gland and pancreas. Histochemistry and immunostaining (by both light and electron microscopy), combined with functional assays, were used to describe the localization patterns and enzyme activities in the organs of wild-type and NTPDase1/cd39-null mice. Pancreatic acinar cells and salivary gland acinar/myoepithelial cells were positive for NTPDase1 and NTPDase2. Ecto-ATPase activity was slightly higher in salivary glands. Ductal epithelial cells expressed ecto-ATPase activity but NTPDase1 and NTPDase2 expression were weak at best. ATPase activity was found in blood vessels of both tissues and its localization pattern overlapped with NTPDase1 staining. In these structures, NTPDase2 antibodies stained the basolateral aspect of endothelial cells and the supporting cells. Biochemical assays and histochemical staining showed relatively high levels of ATPase activity in both glands of cd39(-/-) mice. Our data therefore support a physiological role for NTPDase2 and other ectonucleotidases in the pancreas and salivary glands. Because NTPDase1 is expressed in non-vascular cell types, this finding suggests that NTPDase1 may have functions in the gastrointestinal tract that differ from those demonstrated in the vascular system.     

Requirement of Cys399 for processing of the human ecto-ATPase (NTPDase2) and its implications for determination of the activities of splice variants of the enzyme

Ecto-ATPase (CD39L1) corresponds to the type 2 enzyme of the ecto-nucleoside triphosphate diphosphohydrolase family (E-NTPDase). We have isolated from human ECV304 cells three cDNAs with high homology with members of the E-NTPDase family that encode predicted proteins of 495, 472, and 450 amino acids. Sequencing of a genomic DNA clone confirmed that these three sequences correspond to splice variants of the human ecto-ATPase (NTPDase2 alpha,-2 beta, and -2 gamma). Although all three enzyme forms were expressed heterologously to similar levels in Chinese hamster ovary cells clone K-1 (CHO-K1) cells, only the 495-amino acid protein (NTPDase2 alpha exhibited ecto-ATPase activity. Immunolocalization studies demonstrated that NTPDase2 alpha is fully processed and trafficked to the plasma membrane, whereas the NTPDase2 beta and -2 gamma splice variants were retained in not fully glycosylated forms in the endoplasmic reticulum. The potential roles of two highly conserved residues, Cys399 and Asn443, in the activity and cellular trafficking of the ecto-ATPase were examined. Mutation of Cys399, which is absent in NTPDase2 beta and -2 gamma, produced a protein completely devoid of nucleotidase activity, while mutation of Asn443 to Asp resulted in substantial loss of activity. Neither the Cys399 nor Asn443 mutants were fully glycosylated, and both were retained in the endoplasmic reticulum. These results indicate that the lack of ecto-nucleotidase activity exhibited by NTPDase2 beta and -2 gamma and the C399S mutant, as well as the large reduction of activity in the N443D mutant are due to alterations in the folding/maturation of these proteins.     

The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'apyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.     

Either the carboxyl- or the amino-terminal region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8)

Human ecto-ATPase (E-NTPDase 2) and chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8) are cell surface nucleotidases with two transmembranous domains, one each at the N- and C-termini. Hydrolysis of substrates occurs in active sites residing in their extracellular domains. Human ecto-ATPase activity is decreased by NP-40 and at temperatures higher than 37 degrees C. Reduction of activity is abolished by prior cross-linking of the ecto-ATPase by lectin and chemical cross-linking agents [Knowles, A. F., and Chiang, W.-C. (2003) Arch. Biochem. Biophys. 418, 217-227]. In contrast, the chicken ecto-ATP-diphosphohydrolase is not inhibited by NP-40, and activity is approximately 2-fold higher at 55 degrees C. To determine if the transmembranous domains of the two E-NTPDases mediate their respective responses to detergents and high temperature, we first constructed a chimera (ck-hu ACR5) in which the C-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase. While this chimera displays many similar enzymatic characteristics as the parental chicken ecto-ATP-diphosphohydrolase, its inhibition by NP-40, high temperature, and substrate resemble that of the human ecto-ATPase, which donates the C-terminus including the C-terminal transmembranous domain. Additionally, comparison of the effects of ConA, disuccinimidyl suberate, and glutaraldehyde on the parental enzymes and the chimera indicated that catalysis which occurs in the extracellular domains of the two E-NTPDases responds differently to conformational constraints. Enzyme activity of a second chimera (ck-hu ACR1) in which the N-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase is also inhibited by NP-40 and is less active at 55 degrees C; however, its temperature dependence differs from that of ck-hu ACR5. These results indicate that (1) the C- and N-termini of the two E-NTPDases encompassing the two transmembranous domains are important elements in determining the sensitivity of the human ecto-ATPase to NP-40 and high temperatures; (2) incorporation of either the C- or N-terminus of the human ecto-ATPase alone in the chicken ecto-ATP-diphosphohydrolase is sufficient to impart negative regulation on ATP hydrolysis due to membrane perturbation; and (3) interactions of the two sets of heterologous transmembranous domains are not equivalent, which are most likely related to their different amino acid sequences.     

Role of the conserved lysine within the Walker A motif of human DMC1

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1_K132R) or alanine (hDMC1_K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1_K132R and hDMC1_K132A variants were devoid of ATP hydrolysis. The hDMC1_K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1_K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1_K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.     

A high-resolution genetic, physical, and comparative gene map of the doublefoot (Dbf) region of mouse chromosome 1 and the region of conserved synteny on human chromosome 2q35.

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0.4-cM (+/-0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.     

Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding

Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ecto-nucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported (J. Biol. Chem. (2001) 276, 41518-41525) that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of coexpressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ER-targeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.     

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.     

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.     

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

The untranslated region of exon 2 of the human neuronal nitric oxide synthase (NOS1) gene exerts regulatory activity

Expressional dysregulation of the human neuronal nitric oxide synthase (NOS1) gene represents an important mechanism in the pathogenesis of certain neuronal disease states. The structure and regulation of the human NOS1 gene is highly complex based on cell type- and stimulus-dependent usage of multiple exon 1 variants. Here we demonstrate that the untranslated region of exon 2 exerts promoter and enhancer functions as well, facilitated in large part by cooperative interaction of two conserved adjacent CREB/AP-1 binding sites. In human neuronal A673 cells, NOS1 expression is stimulated by several compounds which act through these sites, but also stimulate the combined promoter region of exons 1f and 1g. While stimulation of NOS1 expression by dibutyryl-cAMP is mediated by protein kinase A (blocked by H-89), the antiepileptic drug valproic acid is likely to activate phosphatidylinositol-3 kinase (inhibited by LY 294002).     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging,aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.