However, several independent lines of experimental evidence strongly indicated that non-lysosomal pathways have an important role in intracellular proteolysis, although their identity and mechanisms of action remained obscure. The discovery of the ubiquitin—proteasome system resolved this enigma.
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Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system. USA 78 , — The question that is commonly asked is: Which comes first, the malfunction of the UPS or the accumulation of toxic proteins that inhibit the UPS? Some studies suggest that it can be both Ciechanover and Brundin, For example, in AD brains proteasome activity is decreased due to a reduction in trypsin and chymotrypsin-like activities Lopez-Salon et al.
In this regard, mole rats, which are known to have a maximum lifespan of Therefore, further studies are needed to find novel therapeutic target opportunities focusing on positive regulators of the UPS. The ALP involves the biogenesis of a unique organelle enclosed by a double lipid bilayer, named the autophagosome.
The basal formation of autophagosomes occurs constitutively to regulate cellular homeostasis and different physiological processes Reggiori and Klionsky, ; Mizushima and Levine, ; Deretic et al. Nevertheless, the ALP is highly inducible by environmental changes and stress stimuli, such as nutrient starvation, growth factors, pathogen infection, protein aggregates or damaged organelles, working as a highly dynamic process that can resolve a variety of cellular demands Murrow and Debnath, Autophagosomes engulf cytoplasmic constituents such as protein aggregates and damaged organelles Khaminets et al.
Autophagosomes can also fuse with endosomes forming an intermediate organelle called the amphisome that, in a similar way to autophagosomes, fuses with lysosomes Yi and Tang, ; Fader and Colombo, ; Patel et al. Whether amphisomes represent a mechanism to eliminate damaged endosomes or to eliminate the cargo that is selected in these organelles is still unclear. Moreover, whether endosomes contribute or not to the initial stages of autophagosome formation remains to be elucidated.
Interestingly, depletion of endosomal sorting complexes required for transport ESCRT components—essential machinery for multivesicular body MVB biogenesis—accumulates autophagosomes. Altogether, this strongly suggests that MVBs contribute to the efficient clearance of these aggregates through the ALP.
Nutrient starvation inhibits mTORC1 and activates the ULK1 complex, relocating this kinase to the pre-autophagosomal membrane structure PAS to promote the first steps of autophagosome biogenesis Itakura and Mizushima, Phosphorylated Beclin-1 activates the pro-autophagic PI3K-III complex and enhances the production of a specific pool of phosphatidylinositolphosphate PI3P , a phospholipid that functions as a signal for PI3P effectors, which allows for the recruitment of downstream autophagy core machinery components Lu et al.
One intriguing question in this field is: What is the major membrane source for autophagosome biogenesis? Cell compartments such as the Golgi apparatus van der Vaart and Reggiori, ; Yamamoto et al. Nevertheless, most of the evidence indicates that the endoplasmic reticulum ER is a key organelle that supplies a platform to the PAS.
Studies suggest that the omegasome acts as a platform for the recruitment of specific machineries implicated in the subsequent biogenesis of the isolation membrane or phagophore Axe et al. Moreover, it has been shown that the mammalian small guanosine triphosphate hydrolase GTPase Rab1 and its yeast homolog Ypt1 regulate autophagosome formation Zoppino et al.
These GTPases are normally located in early secretory compartments such as the ER and Golgi apparatus, where their functions in protein trafficking is achieved Plutner et al. In mammalian cells, an earlier report showed that GFP-Rab1B colocalizes with autophagosomes, which increases under nutrient starvation. This data indicates that Rab1 could be considered as an important protein for the initial steps of autophagosome formation, mediating the early recruitment of the autophagy core machinery to the ER and Golgi apparatus.
Supporting this data, a recent study found that Rab1B associates with Atg9A-positive vesicles, an autophagy related Atg transmembrane protein that controls the early steps of autophagosome biogenesis Kakuta et al.
Until now few proteins have been reported as negative regulators of autophagy. Nevertheless, Rubicon RUN domain-containing Beclin1-interacting protein has been described as a suppressor of the later steps of autophagy.
Early studies indicate that Rubicon is localized at endolysosomal structures where it binds to the PI3K-III complex, inhibiting its activity and impairing the maturation of autophagosomes and endolysosomal compartments Zhong et al.
These findings support the idea that Rubicon together with Rab7 must be considered as crucial regulators of autophagy, offering future therapeutic targets of intervention. Autophagosomes require fully active lysosomes to degrade their content. Moreover, in the last 8 years several studies revealed a key contribution of transcriptional activity in the ALP. These results indicate a concerted mechanism to induce expression of lysosomal genes that implicates mTORC1 inactivation and calcineurin activation.
It also suggests that calcium release from intracellular compartments could lead to calcineurin-mediated ALP activation. Amyloid precursor protein APP is a type I transmembrane protein and is a substrate of two alternative proteolytic processing pathways.
An early ultrastructural study showed that dystrophic neurites in human brains with AD are characterized by the accumulation of enlarged autophagosomes, amphisomes and endolysosomal organelles Nixon et al.
Abnormal axonal endolysosomal structures contain decreased levels of luminal hydrolases e. Studies show that in neurons autophagosome biogenesis begins in distal axons and its maturation process occurs along the axon toward the cell body through a mechanism that depends on retrograde transport Maday et al.
Moreover, it has been proposed that autophagosomes fused with endosomes loaded on dynein motors in distal axons undergo retrograde transport towards the soma for content degradation Cheng et al. Interestingly, in AD neurons amphisomes accumulate in axons probably due to a failure in this retrograde pathway. Because of the functional connection between Rab5 and Rab7 Poteryaev et al. This was also observed when the fusion step between autophagosomes and the endolysosomal compartment was disrupted.
Functionally, it has been proposed that UCH-L1 helps to maintain a stable pool of monoubiquitin during the ubiquitylation process Tramutola et al. Moreover, its hydrolase activity is essential for the maintenance of synaptic and cognitive function in mouse brains. The herpes simplex virus type 1 HSV-1 is a ubiquitous neurotropic virus that establishes lifelong latent infections in neurons and is a risk factor for neuronal damage and neurodysfunction in AD Ando et al.
Interestingly, new findings are beginning to reveal that the observed negative effects of HSV-1 neuronal infection could be linked with key players of the ALP Ando et al. Later it was found that ICP Interestingly, ICP The authors found that neuronal HSV-1 infected cells show a massive increase in autophagosomal-like structures, probably due to inefficient fusion of these organelles with lysosomes Santana et al.
Considering that HSV-1 is a neurotropic virus that causes lifelong latent infection in host neurons as well as the abundant literature linking HSV-1 with AD pathogenesis, it would be interesting to explore whether HSV-1 could be linked to abnormal accumulation of autophagosomes in dystrophic neurites from post-mortem AD patients Nixon et al. Autophagosomes sequester these proteins and deliver them to lysosomes for degradation.
The ALP by contrast to the UPS can degrade a much wider spectrum of substrates that tend to be bulkier, such as protein complexes, oligomers and aggregates, and even whole cellular organelles.
Although the UPS and ALP were considered for a long time as independent mechanisms, a growing body of evidence indicates an intimate crosstalk and cooperation between both pathways Korolchuk et al. Additionally, it has been reported that certain enzymes of the ubiquitylation machinery participate in both degradation pathways.
In summary, disturbances in common molecular aspects of these pathways is particularly relevant to pathophysiological conditions that provoke the accumulation of aberrant proteins, such as in aging as well as in a variety of late-onset neurodegenerative disorders, including AD. Under these conditions, any decay in the UPS and ALP will affect normal cellular function and their ability to effectively counteract proteotoxic stressors Vernace et al.
Ubiquitylation for many years has been studied as the universal signal for the elimination of substrates used for degradation systems. While Klinked polyubiquitin chains work as the canonical signal for proteasomal degradation, Klinked polyubiquitin chains are thought to mediate degradation through the ALP Grice and Nathan, In contrast to this hypothesis, recent findings propose that all chain types are capable of facilitating degradation by both pathways, indicating that the key determinant would be based on the monomeric or oligomeric physical states of these receptors rather than on a specific ubiquitin-moiety Lu et al.
In this regard, several ubiquitin receptors that mediate the recognition of ubiquitylated cargoes in the UPS and ALP have been described Cohen-Kaplan et al. Proteasome receptors cannot oligomerize and bind with high affinity to polyubiquitin chains on soluble substrates rather than on aggregates Lu et al.
By contrast, autophagy receptors gain higher affinity for polyubiquitin chains through their oligomerization, allowing for efficient removal of ubiquitylated aggregates and altogether ensuring the most suitable pathway for their degradation Lu et al. Interestingly, oligomerization of ubiquitin receptors is controlled by PTMs, which determine their efficient function Pan et al.
Among all PTMs, phosphorylation and ubiquitylation were recently characterized, showing great potential in proteostasis control. In contrast, ubiquitylation of the K7 residue within its PB1 domain abrogates its oligomerization and its ability to reduce protein aggregates Pan et al.
Altogether this opens another level of complexity to the process, where the regulation of the NBR1 function by PTMs has only recently started to be characterized Nicot et al.
Moreover, how different kinases or DUBs implicated in the control of proteasomal function are affected upon ALP deficiency is completely unexplored. Finally, an increase in protein aggregate levels has been shown to block the UPS by physical interaction Mayer et al. Ubiquilins UBQLNs are proteins that function as ubiquitin receptors regulating the degradation of ubiquitylated proteins in eukaryotes.
Functional analysis demonstrated that mutations in UBQLN2 within its middle region leads to an impairment of protein degradation, linking this protein to defects in the proteostasis of brain tissue Deng et al. In fact, it was recently demonstrated that UBQLN2 facilitates the clearance of protein aggregates through its interaction with the chaperones HSPHSP that facilitate protein disaggregation, shuttling ubiquitylated proteins for degradation through the UPS, a pathway that is functional even in ALP-deficient cells Hjerpe et al.
Altogether, this expands the roles of UBQLNs in protein degradation and opens interesting aspects that could be exploited. Consistent with this, these authors demonstrated reduced levels of UBQLN1 in post mortem frontal cortex samples derived from late onset AD patients. Aggresomes are perinuclear inclusion bodies located close to the microtubule-organizing center MTOC; Kawaguchi et al. They play a protective role when cells face overloading of abnormal or damaged proteins that cannot be efficiently eliminated by the UPS.
Despite the role of Rpn11 in the UPS where it catalyzes the deubiquitylation of the vast majority of the proteins that enter the proteolytic chamber of the 20S proteasome, normally coupled to Klinked polyubiquitin chains, recent findings indicate that Rpn11 also participates in the clearance of protein aggregates via the ALP.
Consistent with these findings, Rpn11 expression was shown to be highly reduced in human AD brain tissue, suggesting that it has a crucial function in neuronal proteostasis Puthiyedth et al. Removal of ubiquitin chains by Rpn11 in aggresomes produces a large number of free unanchored Kubiquitin chains, molecules that surprisingly seem to be critical for coordinating the elimination of protein aggregates through the ALP Hao et al.
Free unanchored Kubiquitin chains activate the non-canonical function of HDAC6, currently known as a histone deacetylase. These chains trigger the interaction of HDAC6 with dynein motors, allowing for the efficient recruitment of protein aggregates and their transport along microtubules redirecting them to the MTOC to form aggresomes Kawaguchi et al.
Moreover, free unanchored Kubiquitin chains are key molecules for efficient autophagosome-lysosome fusion at the MTOC, a key step for final degradation of protein aggregates Hao et al. In agreement with these findings, deficiency of TRIM50 abrogates the clearance of aggresomes Fusco et al.
Therefore, collective data provide evidence regarding the central role of Rpn11 and free unanchored Kubiquitin chains in a cascade of events that regulate aggresome degradation via the ALP.
Further studies will help to define the consequence of Rpn11 inhibition in brain function and possible regulatory mechanisms of its activity. We have discussed the relevance of phosphorylation as a central hallmark in the control of proteostasis. This increases its affinity for polyubiquitin chains and their degradation via the ALP, substrates that would otherwise accumulate due to their poor degradation by the UPS Matsumoto et al.
PKA activation on the other hand, which is known to enhance proteasome activity Asai et al. Finally, p38 MAPK, which is known to mediate a negative regulatory role in proteasome activity is also a negative player in the ALP through different mechanisms. The main goal of numerous studies is to discover pharmacological agents that can enhance the function of the two major catabolic pathways in eukaryotic cells, pathways whose function declines gradually during aging and age-related neurodegenerative diseases.
Therefore, finding compounds that increase proteasome activity is a challenging and ongoing goal in biomedicine. Up to now, few compounds are known to activate proteasome activity in vivo.
Sulforaphane increases proteasome levels in vivo through induction of the transcription factor Nrf2 Liu et al. Together with the proteasome, another well-investigated approach is to enhance the activity of the ALP. Another interesting compound is Curcumin, a polyphenol antioxidant that is highly enriched in yellow curry.
Trehalose, another attractive natural compound that is not synthesized in mammals and is widely used in food, increases the removal of pathogenic aggregates through the enhancement of autophagy Sarkar et al.
Finally, hyperforin and its derivative Tetrahydrohyperforin IDN , an active component of the plant Hypericum perforatum that is better known as St. Emerging evidence of proteasome and ALP activators offers potent therapeutic approaches to prevent or delay symptoms of AD pathogenesis. A current fundamental challenge in biomedicine is the identification of novel approaches to promote activation of both systems to prevent accumulation of protein aggregates and their harmful deleterious effects.
One interesting option is the phosphorylation of key proteins implicated in these pathways Figure 1. Another interesting target is Rpn11, which releases free unanchored ubiquitin chains, a key step for protein entry into the proteolytic chamber of the proteasome Figure 1.
Interestingly, free unanchored ubiquitin chains are also crucial for the degradation of protein aggregates by the ALP. Recently it was reported that Rpn11 can be acetylated, ubiquitylated and phosphorylated 1 , however, the biological function of these PTMs has not been studied. It would be of great interest to evaluate whether any of these modifications increase Rpn11 activity. Figure 1. Autophagy-lysosomal pathway ALP and ubiquitin-proteasome system UPS pathways under normal and pathological conditions.
Proteins are tagged with ubiquitin conjugates through a sequential enzymatic mechanism involving three classes of enzymes, E1, E2 and E3. Under normal conditions , ubiquitylated substrates are recognized by ubiquitin receptors present in ALP and UPS pathways and efficiently eliminated. In the UPS, substrates are subsequently deubiquitylated by RPN11, a key step for substrate degradation and amino acid recycling. Ubiquitin receptors in the ALP, in contrast to the UPS, form oligomers to facilitate substrate recognition and autophagosomal recruitment.
Altogether, leading to the accumulation of deleterious protein aggregates. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The proteasome is a cylindrical protein complex found in the cytosol which cleaves up proteins tagged with ubiquitin. To accomplish this, an E1 enzyme activates a ubiquitin molecule, transfers it to an E2 enzyme, and finally an E3 ubiquitin ligase covalently attaches ubiquitin to a lysine K on the protein to be degraded.
There are a huge variety of different E3 ubiquitin ligases, reflecting the many different regulatory pathways by which the cell selects and recognizes proteins it wants to flag for degradation.
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