In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Targeted protein degradation (TPD) is an emerging therapeutic modality with the potential to tackle disease-causing proteins that have historically been highly challenging to target with conventional small molecules. In the 20 years since the concept of a proteolysis-targeting chimera (PROTAC) molecule harnessing the ubiquitin–proteasome system to degrade a target protein was reported, TPD has moved from academia to industry, where numerous companies have disclosed programmes in preclinical and early clinical development. With clinical proof-of-concept for PROTAC molecules against two well-established cancer targets provided in 2020, the field is poised to pursue targets that were previously considered ‘undruggable’. In this Review, we summarize the first two decades of PROTAC discovery and assess the current landscape, with a focus on industry activity. We then discuss key areas for the future of TPD, including establishing the target classes for which TPD is most suitable, expanding the use of ubiquitin ligases to enable precision medicine and extending the modality beyond oncology. Targeted protein degradation with proteolysis-targeting chimeras (PROTACs) has the potential to tackle disease-causing proteins that have historically been highly challenging to target with conventional small molecules. This article summarizes the first two decades of PROTAC discovery and discusses key areas for the future of this therapeutic modality, including establishing the target classes for which it is most suitable and extending its application beyond oncology. 

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PROTAC targeted protein degraders: the past is prologue

Autophagy in Human Diseases Autophagy is a complex process of intracellular degradation of senescent or malfunctioning organelles. Dysregulated autophagy is associated with certain cancers, neurode...

Autophagy in Human Diseases

Autophagy is a process in which intracellular components and dysfunctional organelles are delivered to the lysosome for degradation and recycling. Autophagy has various connections to a large number of human diseases, as its functions are essential for cell survival, bioenergetic homeostasis, organism development, and cell death regulation. In the past two decades, substantial effort has been made to identify the roles of autophagy in tumor suppression and promotion, neurodegenerative disorders, and other pathophysiologies. This review summarizes the current advances and discusses the unanswered questions in understanding the involvement of autophagy in pathogenic mechanisms of disease, primarily focusing on cancer and neurodegenerative diseases.

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Autophagy and disease: unanswered questions.

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation. Lysosome-targeting chimeras (LYTACs) based on glycan ligands of the asialoglycoprotein receptor facilitate the cell-specific targeting and turnover of proteins by lysosomal enzymes, expanding the scope of LYTAC-mediated targeted protein degradation.

LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington’s disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington’s disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds. Compounds that interact with mutant huntingtin and an autophagosomal protein are able to reduce cellular levels of mutant huntingtin by targeting it for autophagic degradation, demonstrating an approach that may have potential for treating proteopathies.

/pdf/allele-selective-lowering-of-mutant-htt-protein-by-htt-lc3-3x806r385o.pdf

Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds

Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders, including Huntington disease [caused by expanded CAG repeats (CAGr) in the HTT gene], and amyotrophic lateral sclerosis [ALS, possibly caused by expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene], of which the molecular mechanisms remain unclear. Here, we demonstrated that lowering the Drosophila homologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and the shortened life-span in Drosophila expressing expanded CAGr or expanded G4C2r. Expression of human tau (htau4R) restored the disease-related phenotypes that had been mitigated by the loss of dtau, suggesting an evolutionarily-conserved role of tau in neurodegeneration. We further revealed that G4C2r expression increased tau accumulation by inhibiting autophagosome-lysosome fusion, possibly due to lowering the level of BAG3, a regulator of autophagy and tau. Taken together, our results reveal a novel mechanism by which expanded G4C2r causes neurodegeneration via an evolutionarily-conserved mechanism. Our findings provide novel autophagy-related mechanistic insights into C9ORF72-ALS and possible entry points to disease treatment.

/pdf/tau-accumulation-via-reduced-autophagy-mediates-ggggcc-1jh5o5vm6w.pdf

Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS

Huntington's Disease (HD) is a monogenetic neurodegenerative disorder mainly caused by the cytotoxicity of the mutant HTT protein (mHTT) encoded by the mutant HTT gene. Lowering HTT mRNA has been extensively studied as a potential therapeutic strategy, but how its level is regulated endogenously has been unclear. Here we report that the RNA-binding protein (RBP) HuR interacts with and stabilizes HTT mRNA in an mHTT-dependent manner. In HD cells but not wild-type cells, siRNA knockdown or CRISPR-induced heterozygous knockout of HuR decreased HTT mRNA stability. HuR interacted with HTT mRNA at a conserved site in exon 11 rather than the 3'-UTR region of the mRNA. Interestingly, this interaction was dependent on the presence of mHTT, likely via the activation of MAPK11, which enhanced cytosolic localization of the HuR protein. Thus, mHTT, MAPK11 and HuR may form a positive feedback loop that stabilizes HTT mRNA and enhances mHTT accumulation, which may contribute to HD progression. Our data reveal a novel regulatory mechanism of HTT mRNA via non-canonical binding of HuR.

HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner.

Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders including Huntington disease (HD, caused by the expanded CAG repeats (CAGr) in the HTT gene) and amyotrophic lateral sclerosis (ALS, could be caused by the expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene), of which the molecular mechanisms remain unclear. Here we demonstrate that loss of the Drosophila orthologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and shortened life-span in Drosophila models expressing mutant HTT protein with expanded CAGr or the expanded G4C2r. Importantly, expression of human tau (htau4R) restored the disease-relevant phenotypes that were mitigated by the loss of dtau, suggesting a conserved role of tau in neurodegeneration. We further discovered that G4C2r expression increased tau accumulation, possibly due to reduced activity of BAG3-mediated autophagy. Our study reveals a conserved role of tau in G4C2r-induced neurotoxicity in Drosophila models, providing mechanistic insights and potential therapeutic targets.

https://www.biorxiv.org/content/biorxiv/early/2019/08/07/727008.full.pdf

Yimin Sun

Papers

Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds

Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS

HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner.

Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration