The proteasome and its role in the degradation of oxidized proteins

The generation of free radicals and the resulting oxidative modification of cell structures are omnipresent in mammalian cells. This includes the permanent oxidation of proteins leading to the disruption of the protein structure and an impaired functionality. In consequence, these oxidized proteins have to be removed in order to prevent serious metabolic disturbances. The most important cellular proteolytic system responsible for the removal of oxidized proteins is the proteasomal system. For normal functioning, the proteasomal system needs the coordinated interaction of numerous components. This review describes the fundamental functions of the 20S "core" proteasome, its regulators, and the roles of the proteasomal system beyond the removal of oxidized proteins in mammalian cells.     

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The "quality control" compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the "aggresomes."     

Quantitative analysis of prion-protein degradation by constitutive and immuno-20S proteasomes indicates differences correlated with disease susceptibility

The main part of cytosolic protein degradation depends on the ubiquitin-proteasome system. Proteasomes degrade their substrates into small peptide fragments, some of which are translocated into the endoplasmatic reticulum and loaded onto MHC class I molecules, which are then transported to the cell surface for inspection by CTL. A reliable prediction of proteasomal cleavages in a given protein for the identification of CTL epitopes would benefit immensely from additional cleavage data for the training of prediction algorithms. To increase the knowledge about proteasomal specificity and to gain more insight into the relation of proteasomal activity and susceptibility to prion disease, we digested sheep prion protein with human constitutive and immuno-20S proteasomes. All fragments generated in the digest were quantified. Our results underline the different cleavage specificities of constitutive and immunoproteasomes and provide data for the training of prediction programs for proteasomal cleavages. Furthermore, the kinetic analysis of proteasomal digestion of two different alleles of prion protein shows that even small changes in a protein sequence can affect the overall efficiency of proteasomal processing and thus provides more insight into the possible molecular background of allelic variations and the pathogenicity of prion proteins.     

Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.     

Protection of Extraribosomal RPL13a by GAPDH and Dysregulation by S-Nitrosylation

Multiple eukaryotic ribosomal proteins (RPs) are co-opted for extraribosomal "moonlighting" activities, but paradoxically, RPs exhibit rapid turnover when not ribosome-bound. In one illustrative case of a functional extraribosomal RP, interferon (IFN)-γ induces ribosome release of L13a and assembly into the IFN-gamma-activated inhibitor of translation (GAIT) complex for translational control of a subset of?inflammation-related proteins. Here we show GAPDH functions as a chaperone, shielding newly released L13a from proteasomal degradation. However, GAPDH protective activity is lost following cell?treatment with oxidatively modified low density lipoprotein and IFN-γ. These agonists stimulate S-nitrosylation at Cys(247) of GAPDH, which fails to interact with L13a, causing proteasomal degradation of essentially the entire cell complement of L13a and defective translational control. Evolution of extraribosomal RP activities might require coevolution of?protective chaperones, and pathological disruption of either protein, or their interaction, presents an alternative mechanism of diseases due to RP defects, and targets for therapeutic intervention.     

Peptides that activate the 20S proteasome by gate opening increased oxidized protein removal and reduced protein aggregation

The proteasome is a multicatalytic protease that is responsible for the degradation of the majority of intracellular proteins. Its role is correlated with several major regulatory pathways that are involved in cell cycle control, signaling, and antigen presentation, as well as in the removal of oxidatively damaged proteins. Although several proteasomal catalytic inhibitors have been described, very few activators have been reported to date. Some reports in the literature highlight the cellular protective effects of proteasome activation against oxidative stress and its effect on increased life span. In this work, we describe a peptide named proteasome-activating peptide 1 (PAP1), which increases the chymotrypsin-like proteasomal catalytic activity and, consequently, proteolytic rates both in vitro and in culture. PAP1 proteasomal activation is mediated by the opening of the proteasomal catalytic chamber. We also demonstrate that the observed proteasomal activation protected cells from oxidative stress; further, PAP1 prevented protein aggregation in a cellular model of amyotrophic lateral sclerosis. The role of 20SPT gate opening underlying protection against oxidative stress was also explored in yeast cells. The present data indicate the importance of proteasomal activators as potential drugs for the treatment of pathologies associated with the impaired removal of damaged proteins, which is observed in many neurodegenerative diseases.     

The complexity of recognition of ubiquitinated substrates by the 26S proteasome

The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by – at that time – an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Ubiquitin versus misfolding: The minimal requirements for inclusion body formation

Ubiquitin-containing inclusion bodies are characteristic features of numerous neurodegenerative diseases, but whether ubiquitin plays a functional role in the formation of these protein deposits is unclear. In this issue, Bersuker et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511024) report that protein misfolding without ubiquitylation is sufficient for translocation into inclusion bodies.A large number of sporadic and familial neurodegenerative diseases that differ in their age of onset and manifestation share striking pathological features at the cellular level, suggesting that a common etiology may be responsible for the demise of neurons. Most notable is the aggregation of improperly folded proteins in affected neurons in these so-called protein misfolding diseases that include Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob disease, as well as amyotrophic lateral sclerosis and other motor neuron diseases. Protein aggregates are inherently toxic for cells, underscoring their candidate status as a common denominator in these diseases (Bucciantini et al., 2002). A causative role for aberrant protein conformations is further strengthened by the existence of a family of rare, inheritable neurodegenerative disorders, which are a direct consequence of expansions of polyglutamine repeats that render the mutant proteins prone to aggregation. Given that neuronal cells often must last an organism’s lifetime with little opportunity to dilute protein waste through cell division, it is not hard to imagine that they are particularly susceptible to the gradual accumulation of aberrant proteins that favor precipitation in insoluble protein aggregates.Neurons and other cells have three major lines of defense to minimize the damage that aggregation-prone proteins can cause to cellular homeostasis (Fig. 1). The first two are based on a seek-and-destroy strategy in which the two main intracellular proteolytic systems play complementary roles. Although monomeric aberrant proteins are efficiently targeted for hydrolysis in proteasomes, these proteolytic complexes are unable to process oligomeric protein aggregates (Verhoef et al., 2002). Destruction of proteins in proteasomes requires complete unfolding of the deemed proteins, which may be hard, if not impossible, in the case of tightly associated misfolded proteins. Macroautophagy, however, is a proteolytic pathway that is able to process oligomeric misfolded proteins, as it involves the capturing of cytosolic constituents, including macromolecular complexes like protein aggregates, in double-membrane vesicles that fuse with lysosomes (Ravikumar et al., 2004). As such, macroautophagy complements proteasomal degradation in keeping the cellular environment free from toxic protein species.Open in a separate windowFigure 1.Three lines of defense against misfolded proteins. There are three protective mechanisms that are involved in minimizing the toxicity of misfolded proteins: proteasomal degradation (I), macroautophagic clearance (II), and inclusion body formation (III). Ubiquitin is linked to each of these processes, as it can target proteins for proteasomal and macroautophagosomal degradation and is enriched in inclusion bodies.In the unfortunate case that the production of aggregation-prone proteins exceeds the capacity of both the proteasomal and lysosomal systems, a potential catastrophic situation arises as misfolded proteins may precipitate in large, insoluble aggregates. In these cases, a third protective mechanism can come to the rescue and primarily provides damage control, as it intercepts protein aggregates and sequesters them in dedicated subcellular structures, thereby minimizing the harm that the aberrant proteins may cause (Johnston et al., 1998). It is this process that is responsible for the formation of the characteristic inclusion bodies that are typically observed in affected neurons and known under different names depending on the neurodegenerative disorder in which they occur, such as Lewy bodies in Parkinson’s disease, Bunina bodies in amyotrophic lateral sclerosis, and intranuclear inclusions in several polyglutamine disorders (Alves-Rodrigues et al., 1998). Although these structures were originally considered as a potential cause for the cellular pathology, a large body of evidence suggests that they actually lessen the cellular damage caused by toxic proteins (Arrasate et al., 2004). Although their presence may not be without negative consequence for the cells, the controlled formation of waste deposits may be the best possible option for the cell when facing excessive amounts of aggregated proteins.Interestingly, the protein modifier ubiquitin, a posttranslational modification covalently linked to lysine residues of target proteins, appears to be somehow involved in each of these three protective mechanisms. Polyubiquitylation, or conjugation of a chain of ubiquitin molecules, targets proteins for proteasomal degradation and is likewise also critical for proteasomal destruction of misfolded proteins (Kleiger and Mayor, 2014). Even though macroautophagy was originally seen as a nonselective catabolic pathway, more recent studies have suggested that it also involves a high level of specificity with ubiquitin chains being an important substrate recruitment signal (Kraft et al., 2010). In sharp contrast to the well-defined targeting function of ubiquitin in these proteolytic mechanisms, its possible role in the formation of inclusion bodies has been less clear. This is somewhat ironic, given that the initial observations of ubiquitin-positive inclusions in neurodegeneration date back almost three decades (Mori et al., 1987) and have been among the main findings that sparked the interest in a possible role of dysfunctional ubiquitin-dependent proteasomal degradation in neurodegenerative disorders (Cummings et al., 1998). In this issue, Bersuker et al. revisited this important question using an elegant system that allowed them to follow specifically designed reporter proteins that could be switched from folded to misfolded states by administration of cell-permeable ligands. Using this approach, they confirmed that introducing a misfolded state resulted in rapid clearance of the reporter proteins by ubiquitin-dependent proteasomal degradation, the first line of defense against misfolded proteins. Consistent with the prevailing model, they also found that the misfolded reporters accumulated in inclusion bodies when they increased the load of aggregation-prone proteins by simultaneously expressing a fragment of mutant huntingtin containing an expanded polyglutamine repeat, the protein responsible for Huntington’s disease. Interestingly, chemical inhibition of the ubiquitin activase, an enzyme that is critical for ubiquitin conjugation, showed that translocation of the reporter proteins to inclusion bodies did not require ubiquitylation, arguing that the misfolded state is sufficient to reach the final destination.If ubiquitin is not needed for targeting misfolded proteins to inclusion bodies, why then do these proteinaceous deposits contain such large amounts of ubiquitin? The fact that ubiquitin is not required for the recruitment of misfolded proteins to inclusion bodies does not exclude the possibility that ubiquitylation targets properly folded proteins to inclusion bodies. Thus, a possible scenario is that inclusion bodies, once they have been seeded by the ubiquitin-independent sequestration of misfolded proteins, will start to gather soluble polyubiquitylated proteins that typically accumulate under conditions of disturbed protein homeostasis. The authors investigated this possibility by expressing a reporter substrate that contained a degradation signal and was therefore efficiently targeted for ubiquitin-dependent proteasomal degradation. Interestingly, even though these substrates accumulated in a ubiquitylated form when proteasomal degradation was obstructed, they did not localize to the inclusion bodies that otherwise gathered misfolded reporters. This suggests that ubiquitin chains—at least those that target substrates for proteasomal degradation—are not sufficient to autonomously target proteins to inclusion bodies and, at the same time, excludes the possibility that their presence is due to a general sequestration of ubiquitylated proteasome substrates.Alternatively, ubiquitin in inclusions may reflect an attempt of the cell to get rid of the sequestrated protein aggregates once they have reached the inclusion body by targeting them for destruction via ubiquitin-dependent proteolytic systems. Indeed, in vivo studies suggest that inclusion bodies are not a dead-end product but can be cleared from affected neurons (Yamamoto et al., 2000). Even though ubiquitin-dependent autophagosomal and proteasomal degradation are primary candidates for facilitating disposal of inclusions (Martín-Aparicio et al., 2001; Wong et al., 2008), it should be noted that it is presently unclear how this would be mechanistically executed. The data presented by Bersuker et al. (2016) show that the pool of ubiquitin in inclusion bodies is rather static, arguing against a direct role in the turnover, if any, of the ubiquitylated proteins present in the inclusions.Where do these findings leave us? It is fair to say that the functional significance of ubiquitin in inclusion bodies remains somewhat elusive. Following the road of exclusion as in the present study, we can put a solid strike through several trivial explanations for the presence of ubiquitin in inclusions, but further research will be needed to get a more definitive answer about ubiquitin’s role in this process or the lack thereof. It also brings up questions about the role of the microtubule-associated deacetylase HDAC6 in this process. Some studies have provided data that support an essential role for this cytosolic deacetylase in transporting aggregates to inclusions by virtue of its ability to simultaneously bind ubiquitin conjugates and the dynein motors that are required for their sequestration (Kawaguchi et al., 2003; Olzmann et al., 2007). However, HDAC6 has also been linked to degradation of aggregation-prone proteins by macroautophagy, suggesting that it may indirectly influence the kinetics of inclusion body formation (Pandey et al., 2007; Lee et al., 2010). Even though these processes are not mutually exclusive and may well be functionally linked, the present findings motivate a closer look at the molecular mechanisms that link HDAC6 to the formation of inclusion bodies. It should be noted that although the presented data demonstrate that the canonical ubiquitin chains that target proteins for proteasomal degradation are insufficient to promote their translocation to inclusion bodies, it does not exclude implication of alternative ubiquitin chains. Ubiquitin modifications come in many different flavors, and, in particular, the K63-linked polyubiquitin chains, which do not target for proteasomal degradation, have been linked to both macroautophagy and inclusion body formation (Lim and Lim, 2011).The present work also underscores the importance of the exclusive role of protein aggregation in directing misfolded proteins to inclusion bodies. This finding resonates with an earlier study from the same group, in which they reported that targeting of misfolded proteins for autophagy is a direct consequence of their aggregation and does not necessarily require ubiquitylation (Riley et al., 2010). A picture starts to emerge of a general strategy in which the attention of these protective mechanisms is directly drawn to the problematic proteins by the very same virtue that causes their misbehavior, namely their tendency to aggregate. The central role of protein aggregation, as opposed to ubiquitylation, may also be relevant for the similarities and dissimilarities between the formation of inclusion bodies in the cytosolic and nuclear compartments of cells. Whereas the present study probes into the role of ubiquitin in the generation of cytosolic inclusions, intranuclear inclusions are most notoriously associated with the pathology of neurodegenerative diseases. Even though there are fundamental differences in ubiquitin targeting and transport mechanisms between these compartments, the intrinsic property of the proteins to aggregate applies to both, and it is also feasible that in the nucleus, the misfolded domains suffice to facilitate their translocation to inclusion bodies. The lack of a need for a middleman in this critical process may reflect the archaic nature of this innate response and allow rapid incapacitation of these inherently toxic species. This will also ensure that handling of these proteins is not susceptible to disturbed ubiquitin homeostasis, as often is the case in neurodegenerative disorders.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

The Archaeal Proteasome Is Regulated by a Network of AAA ATPases

The proteasome is the central machinery for targeted protein degradation in archaea, Actinobacteria, and eukaryotes. In its basic form, it consists of a regulatory ATPase complex and a proteolytic core particle. The interaction between the two is governed by an HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) at the C terminus of the ATPase subunits, which stimulates gate opening of the proteasomal α-subunits. In archaea, the proteasome-interacting motif is not only found in canonical proteasome-activating nucleotidases of the PAN/ARC/Rpt group, which are absent in major archaeal lineages, but also in proteins of the CDC48/p97/VAT and AMA groups, suggesting a regulatory network of proteasomal ATPases. Indeed, Thermoplasma acidophilum, which lacks PAN, encodes one CDC48 protein that interacts with the 20S proteasome and activates the degradation of model substrates. In contrast, Methanosarcina mazei contains seven AAA proteins, five of which, both PAN proteins, two out of three CDC48 proteins, and the AMA protein, function as proteasomal gatekeepers. The prevalent presence of multiple, distinct proteasomal ATPases in archaea thus results in a network of regulatory ATPases that may widen the substrate spectrum of proteasomal protein degradation.     

The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome

The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.     

N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

Efficient degradation of by‐products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER‐associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER‐phagy receptors for ER‐to‐lysosome‐associated degradation (ERLAD). Demannosylation of N‐linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro‐collagen that cycles of de‐/re‐glucosylation of selected N‐glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD‐resistant misfolded proteins for FAM134B‐driven lysosomal delivery. In summary, we show that mannose and glucose processing of N‐glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.     

FE65 proteins regulate NMDA receptor activation-induced amyloid precursor protein processing

Amyloid precursor protein (APP) family members and their proteolytic products are implicated in normal nervous system function and Alzheimer's disease pathogenesis. APP processing and Aβ secretion are regulated by neuronal activity. Various data suggest that NMDA receptor (NMDAR) activity plays a role in both non-amyloidogenic and amyloidogenic APP processing depending on whether synaptic or extrasynaptic NMDARs are activated, respectively. The APP-interacting FE65 proteins modulate APP trafficking and processing in cell lines, but little is known about their contribution to APP trafficking and processing in neurons, either in vivo or in vitro. In this study, we examined the contribution of the FE65 protein family to APP trafficking and processing in WT and FE65/FE65L1 double knockout neurons under basal conditions and following NMDAR activation. We report that FE65 proteins facilitate neuronal Aβ secretion without affecting APP fast axonal transport to pre-synaptic terminals. In addition, FE65 proteins facilitate an NMDAR-dependent non-amyloidogenic APP processing pathway. Generation of high-molecular weight (HMW) species bearing an APP C-terminal epitope was also observed following NMDAR activation. These HMW species require proteasomal and calpain activities for their accumulation. Recovery of APP polypeptide fragments from electroeluted HMW species having molecular weights consistent with calpain I cleavage of APP suggests that HMW species are complexes formed from APP metabolic products. Our results indicate that the FE65 proteins contribute to physiological APP processing and accumulation of APP metabolic products resulting from NMDAR activation.     

Degradation of nicastrin involves both proteasome and lysosome

The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time- and dose-dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co-immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.