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Macroautophagy/autophagy, an evolutionarily conserved degradation system, serves to clear intracellular components through the lysosomal pathway. Mounting evidence has revealed cytoprotective roles of autophagy; however, the intracellular causes of overactivated autophagy, which has cytotoxic effects, remain elusive. Here we show that sustained proteotoxic stress induced by loss of the RI NG and Ke lch repeat-containing protein C53A5.6/RIKE-1 induces sequestration of LET-363/MTOR complex and overactivation of autophagy, and consequently impairs epithelial integrity in C. elegans . In C53A5.6/RIKE-1-deficient animals, blocking autophagosome formation effectively prevents excessive endosomal degradation, mitigates mislocalization of intestinal membrane components and restores intestinal lumen morphology. However, autophagy inhibition does not affect LET-363/MTOR aggregation in animals with compromised C53A5.6/RIKE-1 function. Improving proteostasis capacity by reducing DAF-2 insulin/IGF1 signaling markedly relieves the aggregation of LET-363/MTOR and alleviates autophagy overactivation, which in turn reverses derailed endosomal trafficking and rescues epithelial morphogenesis defects in C53A5.6/RIKE-1-deficient animals. Hence, our studies reveal that C53A5.6/RIKE-1-mediated proteostasis is critical for maintaining the basal level of autophagy and epithelial integrity. Abbreviations: ACT-5: actin 5; ACTB: actin beta; ALs: autolysosomes; APs: autophagosomes; AJM-1: apical junction molecule; ATG: autophagy related; C. elegans: Caenorhabditis elegans; CPL-1: cathepsin L family; DAF: abnormal dauer formation; DLG-1: Drosophila discs large homolog; ERM-1: ezrin/radixin/moesin; EPG: ectopic P granule; GFP: freen fluorescent protein; HLH-30: helix loop helix; HSP: heat shock protein; LAAT-1: lysosome associated amino acid transporter; LET: lethal; LGG-1: LC3, GABARAP and GATE-16 family; LMP-1: LAMP (lysosome-associated membrane protein) homolog; MTOR: mechanistic target of rapamycin kinase; NUC-1: abnormal nuclease; PEPT-1/OPT-2: Peptide transporter family; PGP-1: P-glycoprotein related; RAB: RAB family; RIKE-1: RING and Kelch repeat-containing protein; SLCF-1: solute carrier family; SQST-1: sequestosome related; SPTL-1: serine palmitoyl transferase family.

Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress. Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; K d , dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D.

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPT P301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis. Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA 1 : bafilomycin A 1 ; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/βIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system.

Cells mainly rely on stress proteins, such as heat-shock proteins (HSPs), to respond to various proteotoxic conditions. These proteins protect tumor cells and enhance their survive. However, the regulation of stress proteins involved in protein quality control (PQC) is still poorly understood. Here, we report that the expression of TRIM11, an important regulator of PQC, is positively correlated with tumor cell surviaval during the proteotoxic conditions induced by anti-tumor drugs. In addition, HSF1 is required for TRIM11-mediated removal of protein aggregates and resistance of proteotoxic stress. During these processes, TRIM11 interacts with and stabilizes HSF1, increaseing HSF1 levels in the nucleus. These findings identify that TRIM11, through cooperation with HSF1, protects cells against the proteotoxic stress and promotes tumor cell survival.

Protein misfolding and protein aggregation are linked to several diseases commonly called as proteinopathies, which include cancer. Understanding the mechanisms of proteostasis could provide newer strategies to combat proteinopathies. We have recently demonstrated a new mechanism where we found that TRIM16 (tripartite motif-containing protein 16) utilizing NRF2-p62 axis and autophagy streamlines the safe disposal of misfolded proteins to maintain protein homeostasis.

The molecular mechanism responsible for cell fate after mitotic slippage remains unclear. We investigated the different postmitotic effects of aneuploidy versus polyploidy using chemical inhibitors of centromere-associated protein-E (CENP-E) and kinesin family member 11 (KIF11, also known as Eg5). Aneuploidy caused substantial proteotoxic stress and DNA damage accompanied by p53-mediated postmitotic apoptosis, whereas polyploidy did not induce these antiproliferative effects.

Although nearly ubiquitous in cancer, aneuploidy exerts detrimental effects on human cells. We recently demonstrated that aneuploid human cells exhibit impaired heat shock factor protein 1 (HSF1) and HSP90 function, suggesting a functional link between two recurring features of cancer cells: aneuploidy and proteotoxic stress. Further, our findings implicate HSF1 as a key factor in mitigating the effects of aneuploidy.

Within tumor cells the heat shock factor 1 (HSF1)-mediated stress response is constitutively mobilized to counter persistent proteotoxic stress, hence sustaining their fragile proteome homeostasis and supporting robust malignant phenotypes. Intriguingly, our new studies reveal that metabolic stressors, such as metformin, inactivate HSF1 and provoke proteomic chaos, thereby impeding tumorigenesis.

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

Mutations in genes that encode tRNAs, aminoacyl-tRNA syntheases, tRNA modifying enzymes and other tRNA interacting partners are associated with neuropathies, cancer, type-II diabetes and hearing loss, but how these mutations cause disease is unclear. We have hypothesized that levels of tRNA decoding error (mistranslation) that do not fully impair embryonic development can accelerate cell degeneration through proteome instability and saturation of the proteostasis network. To test this hypothesis we have induced mistranslation in zebrafish embryos using mutant tRNAs that misincorporate Serine (Ser) at various non-cognate codon sites. Embryo viability was affected and malformations were observed, but a significant proportion of embryos survived by activating the unfolded protein response (UPR), the ubiquitin proteasome pathway (UPP) and downregulating protein biosynthesis. Accumulation of reactive oxygen species (ROS), mitochondrial and nuclear DNA damage and disruption of the mitochondrial network, were also observed, suggesting that mistranslation had a strong negative impact on protein synthesis rate, ER and mitochondrial homeostasis. We postulate that mistranslation promotes gradual cellular degeneration and disease through protein aggregation, mitochondrial dysfunction and genome instability.