Transferrin receptor controls both autophagosome formation and closure via phosphatidylinositol 3-phosphate synthesis.

Autophagosome formation involves multiple sequential steps that need to be coordinated and linked. Here, we describe in mammalian cells that the transferrin receptor (TfR) links LC3 family conjugation to phagophore membranes, an early step in autophagosome biogenesis, with subsequent autophagosome closure. TfR depletion impairs autophagic flux and its overexpression stimulates this catabolic process in an iron-independent manner.

Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson's disease and Huntington's disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling.

SMER28 binding to VCP/p97 enhances both autophagic and proteasomal neurotoxic protein clearance.

The ability to maintain a functional proteome by clearing damaged or misfolded proteins is critical for cell survival, and aggregate-prone proteins accumulate in many neurodegenerative diseases, such as Huntington, Alzheimer, and Parkinson diseases. The removal of such proteins is mainly mediated by the ubiquitin-proteasome system and autophagy, and the activity of these systems declines in disease or with age. We recently found that targeting VCP/p97 with compounds like SMER28 enhances macroautophagy/autophagy flux mediated by the increased activity of the PtdIns3K complex I.

Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance.

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome.

The different autophagy degradation pathways and neurodegeneration.

The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways.

Transient siRNA-mediated protein knockdown in mouse followed by feeding/starving cycle and liver tissue analysis.

We present a protocol for in vivo siRNA-mediated knockdown of a gene of interest in mouse liver using systemic delivery via intravenous injection. We describe a step-by-step protocol for delivery of siRNA particles, with tips on how to optimize dosage. We detail steps for feeding/starving cycles as well as for liver tissue isolation, followed by gene expression analysis, measured at the mRNA and protein levels.

VCP/p97 modulates PtdIns3P production and autophagy initiation.

VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex.

VCP/p97 regulates Beclin-1-dependent autophagy initiation.

Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1.

mTORC2 Assembly Is Regulated by USP9X-Mediated Deubiquitination of RICTOR.

The mechanistic target of rapamycin complex 2 (mTORC2) controls cell metabolism and survival in response to environmental inputs. Dysregulation of mTORC2 signaling has been linked to diverse human diseases, including cancer and metabolic disorders, highlighting the importance of a tightly controlled mTORC2. While mTORC2 assembly is a critical determinant of its activity, the factors regulating this event are not well understood, and it is unclear whether this process is regulated by growth factors.

Erratum: Author Correction: Mendelian neurodegenerative disease genes involved in autophagy.

[This corrects the article DOI: 10.1038/s41421-020-0158-y.].

Mendelian neurodegenerative disease genes involved in autophagy.

The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases.

Non-canonical translation initiation in yeast generates a cryptic pool of mitochondrial proteins.

Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria.

Correction to: Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.

Reference 45 was incorrect in the original version of this article.

Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.

Autophagy is a conserved intracellular degradation pathway essential for protein homeostasis, survival and development. Defects in autophagic pathways have been connected to a variety of human diseases, including cancer and neurodegeneration. In the process of macroautophagy, cytoplasmic cargo is enclosed in a double-membrane structure and fused to the lysosome to allow for digestion and recycling of material.

Quantitative proteomics identifies redox switches for global translation modulation by mitochondrially produced reactive oxygen species.

The generation of reactive oxygen species (ROS) is inevitably linked to life. However, the precise role of ROS in signalling and specific targets is largely unknown. We perform a global proteomic analysis to delineate the yeast redoxome to a depth of more than 4,300 unique cysteine residues in over 2,200 proteins.

The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly.

Disulfide bond formation is crucial for the biogenesis and structure of many proteins that are localized in the intermembrane space of mitochondria. The importance of disulfide bond formation within mitochondrial proteins was extended beyond soluble intermembrane space proteins. Tim22, a membrane protein and core component of the mitochondrial translocase TIM22, forms an intramolecular disulfide bond in yeast.

Chatty Mitochondria: Keeping Balance in Cellular Protein Homeostasis.

Mitochondria are multifunctional cellular organelles that host many biochemical pathways including oxidative phosphorylation (OXPHOS). Defective mitochondria pose a threat to cellular homeostasis and compensatory responses exist to curtail the source of stress and/or its consequences. The mitochondrial proteome comprises proteins encoded by the nuclear and mitochondrial genomes.

Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol.

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death.

Presequence recognition by the tom40 channel contributes to precursor translocation into the mitochondrial matrix.

More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined.

Mia40 and MINOS act in parallel with Ccs1 in the biogenesis of mitochondrial Sod1.

Superoxide dismutase 1 (Sod1) is a major superoxide-scavenging enzyme in the eukaryotic cell, and is localized in the cytosol and intermembrane space of mitochondria. Sod1 requires its specific chaperone Ccs1 and disulfide bond formation in order to be retained in the intermembrane space. Our study identified a pool of Sod1 that is present in the reduced state in mitochondria that lack Ccs1.

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria.

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins.