Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

Using a Modified Proximity Ligation Protocol to Study the Interaction Between Chaperones and Associated Proteins.

Molecular chaperones can interact with multiple proteins to form large networks. Understanding these interactions may shed light on the complexity of the chaperone functions. Here we developed a protocol for a modified proximity ligation-based methodology (PLA) for the detection of protein-protein interactions in order to understand how the Hsp70-Bag3 complex interacts with components of the Hippo signaling pathway. These experiments helped to elucidate the mechanisms of transmission of the proteotoxic stress signal to the Hippo pathway. The modified PLA technology has many advantages compared to co-immunoprecipitation protocols. It has higher sensitivity, is quantitative, and can be done in a 96-well format.

The Hsp70-Bag3 complex modulates the phosphorylation and nuclear translocation of Hippo pathway protein Yap.

Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70-Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.

Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation.

Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligomers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70-Bag3 complex therefore functions as an important signaling node that senses proteotoxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation.

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

Proteasome failure promotes positioning of lysosomes around the aggresome via local block of microtubule-dependent transport.

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species.

A novel approach to recovery of function of mutant proteins by slowing down translation.

Protein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.

The heat shock transcription factor Hsf1 is downregulated in DNA damage-associated senescence, contributing to the maintenance of senescence phenotype.

Heat shock response (HSR) that protects cells from proteotoxic stresses is downregulated in aging, as well as upon replicative senescence of cells in culture. Here we demonstrate that HSR is suppressed in fibroblasts from the patients with segmental progerioid Werner Syndrome, which undergo premature senescence. Similar suppression of HSR was seen in normal fibroblasts, which underwent senescence in response to DNA damaging treatments. The major DNA-damage-induced signaling (DDS) pathways p53-p21 and p38-NF-kB-SASP contributed to the HSR suppression. The HSR suppression was associated with inhibition of both activity and transcription of the heat shock transcription factor Hsf1. This inhibition in large part resulted from the downregulation of SIRT1, which in turn was because of decrease in the expression of the translation regulator HuR. Importantly, we uncovered a positive feedback regulation, where suppression of Hsf1 further activates the p38-NF-κB-SASP pathway, which in turn promotes senescence. Overexpression of Hsf1 inhibited the p38-NFκB-SASP pathway and partially relieved senescence. Therefore, downregulation of Hsf1 plays an important role in the development or in the maintenance of DNA damage signaling-induced cell senescence.

Association of translation factor eEF1A with defective ribosomal products generates a signal for aggresome formation.

Aggresome formation is initiated upon proteasome failure, and facilitates autophagic clearance of protein aggregates to protect cells from proteotoxicity. Here we demonstrate that proteasome inhibition generates a signaling event to trigger aggresome formation. In aggresome signaling, the cell senses a build-up of aberrant newly synthesized proteins. The translation elongation factor eEF1A associated with these species, and knockdown of this factor suppressed aggresome formation. We used the Legionella toxin SidI to distinguish between the function of eEF1A in translation and its novel function in the aggresome formation. In fact, although it strongly inhibited translation, this toxin had only a marginal effect on aggresome formation. Furthermore, SidI reduced the threshold of the aberrant ribosomal products for triggering aggresome formation. Therefore, eEF1A binds defective polypeptides released from ribosomes, which generates a signal that triggers aggresome formation.

Isolation of aggresomes and other large aggregates.

Upon permanent stresses and in various diseases, small protein aggregates may accumulate in cells and cause toxicity. A recently discovered protective system transports these aggregates to the centrosome location via microtubules, to form a large agglomerate of aggregates called the aggresomes. Here, we describe a newly developed method for isolating aggresomes. This principle can also be used for purification of other large structures and even organelles.

Importance of mRNA secondary structural elements for the expression of influenza virus genes.

Development of novel vaccines and therapeutics often requires efficient expression of recombinant viral proteins. Here we show that mutations in essential functional regions of conserved influenza proteins NP and NS1, lead to reduced expression of these genes in vitro. According to in silico analysis, these mRNA regions possess distinct secondary structures sensitive to mutations. We identified a novel structural feature within a region in NS1 mRNA that encodes amino acids essential for NS1 function. Mutations altering this mRNA element lead to significantly reduced protein expression. Conversely, expression was not affected by mutations resulting in amino acid substitutions, when they were designed to preserve this secondary RNA structural element. Furthermore, altering this structure significantly reduced RNA transcription without affecting mRNA stability. Therefore, distinct internal secondary structures of viral mRNA may be important for viral gene expression. If such elements encode amino acids essential for the protein function, then early selection against mutations in this region will be beneficial for the virus. This might point at yet another mechanism of viral evolution, especially for RNA viruses. Finally, introducing mutations into viral genes while preserving their secondary RNA structure, suggests a new method for the generation of efficiently expressed recombinants of viral proteins.

Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery.

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.

Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1.

Abnormal polypeptides that escape proteasome-dependent degradation and aggregate in cytosol can be transported via microtubules to an aggresome, a recently discovered organelle where aggregated proteins are stored or degraded by autophagy. We used synphilin 1, a protein implicated in Parkinson disease, as a model to study mechanisms of aggresome formation. When expressed in naïve HEK293 cells, synphilin 1 forms multiple small highly mobile aggregates. However, proteasome or Hsp90 inhibition rapidly triggered their translocation into the aggresome, and surprisingly, this response was independent on the expression level of synphilin 1. Therefore, aggresome formation, but not aggregation of synphilin 1, represents a special cellular response to a failure of the proteasome/chaperone machinery. Importantly, translocation to aggresomes required a special aggresome-targeting signal within the sequence of synphilin 1, an ankyrin-like repeat domain. On the other hand, formation of multiple small aggregates required an entirely different segment within synphilin 1, indicating that aggregation and aggresome formation determinants can be separated genetically. Furthermore, substitution of the ankyrin-like repeat in synphilin 1 with an aggresome-targeting signal from huntingtin was sufficient for aggresome formation upon inhibition of the proteasome. Analogously, attachment of the ankyrin-like repeat to a huntingtin fragment lacking its aggresome-targeting signal promoted its transport to aggresomes. These findings indicate the existence of transferable signals that target aggregation-prone polypeptides to aggresomes.

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components.

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency.

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.

Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains.

The cell's failure to refold or break down abnormal polypeptides often leads to their aggregation, which could cause toxicity and various pathologies. Here we investigated cellular factors involved in protein aggregation in yeast and mammalian cells using model polypeptides containing polyglutamine domains. In yeast, a number of mutations affecting the complex responsible for formation of the endocytic vesicle reduced the aggregation. Components of the endocytic complex (EC) Sla1, Sla2, and Pan1 were seen as clusters in the polyglutamine aggregates. These proteins associate with EC at the later stages of its maturation. In contrast, Ede1 and Ent1, the elements of EC at the earlier stages, were not found in the aggregates, suggesting that late ECs are involved in polyglutamine aggregation. Indeed, stabilization of the late complexes by inhibition of actin polymerization enhanced aggregation of polypeptides with polyglutamine domains. Similarly, in mammalian cells, inhibitors of actin polymerization, as well as depletion of a mediator of actin polymerization, Arp2, strongly enhanced the aggregation. In contrast, destabilization of EC by depletion or inhibition of a scaffolding protein N-WASP effectively suppressed the aggregation. Therefore, EC appears to play a pivotal role in aggregation of cytosolic polypeptides with polyglutamine domains in both yeast and mammalian cells.

Characterization of proteins associated with polyglutamine aggregates: a novel approach towards isolation of aggregates from protein conformation disorders.

The common feature of many neurodegenerative diseases is emergence of protein aggregates. Identifying their composition can provide valuable insights into the cellular mechanisms of protein aggregation and neuronal death. No reliable method for identification of the aggregate-associated proteins has been available. Here we describe a method for characterization of protein aggregates based on sedimentation of immunocomplexes without involvement of a solid support. As a model, we used the aggregates formed in yeast by a polyglutamine-containing segment of mutant huntingtin. Sixteen proteins associated with the isolated aggregates were identified with 2-D gel electrophoresis followed by mass spectrometry. We found that the aggregates in cells lacking Rnq1 prion recruited lesser amounts of chaperones than those in the wild-type cells. The method can be utilized for characterization of various types of aggregates, prions and very large protein complexes under mild conditions that preserve associated proteins.

A potent small molecule inhibits polyglutamine aggregation in Huntington's disease neurons and suppresses neurodegeneration in vivo.

Polyglutamine (polyQ) disorders, including Huntington's disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products. In polyQ diseases, the pathology and death of affected neurons are associated with the accumulation of mutant proteins in insoluble aggregates. Several studies implicate polyQ-dependent aggregation as a cause of neurodegeneration in HD, suggesting that inhibition of neuronal polyQ aggregation may be therapeutic in HD patients. We have used a yeast-based high-throughput screening assay to identify small-molecule inhibitors of polyQ aggregation. We validated the effects of four hit compounds in mammalian cell-based models of HD, optimized compound structures for potency, and then tested them in vitro in cultured brain slices from HD transgenic mice. These efforts identified a potent compound (IC50=10 nM) with long-term inhibitory effects on polyQ aggregation in HD neurons. Testing of this compound in a Drosophila HD model showed that it suppresses neurodegeneration in vivo, strongly suggesting an essential role for polyQ aggregation in HD pathology. The aggregation inhibitors identified in this screen represent four primary chemical scaffolds and are strong lead compounds for the development of therapeutics for human polyQ diseases.

Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis.

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.