Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes.

Ubiquitination of non-protein substrates.

The covalent attachment of ubiquitin is a common regulatory mechanism in various proteins. Although it has long been thought that the substrates of ubiquitination are limited to proteins, recent studies have changed this view: ubiquitin can be conjugated to lipids, sugars, and nucleotides. Ubiquitin is linked to these substrates by the action of different classes of ubiquitin ligases that have distinct catalytic mechanisms.

Cell biology of protein-lipid conjugation.

Protein-lipid conjugation is a widespread modification involved in many biological processes. Various lipids, including fatty acids, isoprenoids, sterols, glycosylphosphatidylinositol, sphingolipids, and phospholipids, are covalently linked with proteins. These modifications direct proteins to intracellular membranes through the hydrophobic nature of lipids.

The pH-sensing Rim101 pathway regulates cell size in budding yeast.

Although cell size regulation is crucial for cellular functions in a variety of organisms from bacteria to humans, the underlying mechanisms remain elusive. Here, we identify Rim21, a component of the pH-sensing Rim101 pathway, as a positive regulator of cell size through a flow cytometry-based genome-wide screen of Saccharomyces cerevisiae deletion mutants. We found that mutants defective in the Rim101 pathway were consistently smaller than wildtype cells in the log and stationary phases.

Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines.

Ubiquitin is covalently conjugated to phospholipids as well as proteins; however, ubiquitinated phospholipids are less abundant than free ubiquitin and ubiquitinated proteins. Here, we describe protocols to purify ubiquitinated phospholipids in budding yeast and human cells based on their hydrophobicity. Ubiquitinated phospholipids are purified by Triton X-114 phase partitioning and affinity purification and verified by phospholipase D treatment.

Conjugation of the ubiquitin family proteins to phospholipids.

Conjugation of Atg8-family proteins to phosphatidylethanolamine (PE) is important for autophagosome formation. PE conjugation has been thought to be specific to Atg8 among the ubiquitin-family proteins. However, this dogma has not been experimentally verified.

Ubiquitination of phosphatidylethanolamine in organellar membranes.

The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids.

Silencing the G-protein coupled receptor 3-salt inducible kinase 2 pathway promotes human β cell proliferation.

Loss of pancreatic β cells is the hallmark of type 1 diabetes, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human β cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic β cell proliferation.

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood.

BRD4-mediated repression of p53 is a target for combination therapy in AML.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents.

MDH1 and MPP7 Regulate Autophagy in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is driven by metabolic changes in pancreatic cells caused by oncogenic mutations and dysregulation of p53. PDAC cell lines and PDAC-derived xenografts grow as a result of altered metabolic pathways, changes in stroma, and autophagy. Selective targeting and inhibition of one of these may open avenues for the development of new therapeutic strategies.

Identification and Validation of Novel Autophagy Regulators Using an Endogenous Readout siGENOME Screen.

Autophagy is a highly regulated process, and its deregulation can contribute to various diseases, including cancer, immune diseases, and neurodegenerative disorders. Here we describe the design, protocol, and analysis of an imaging-based high-throughput screen with an endogenous autophagy readout. The screen uses a genome-wide siRNA library to identify autophagy regulators in mammalian cells.

Mannose impairs tumour growth and enhances chemotherapy.

It is now well established that tumours undergo changes in cellular metabolism. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy.

Emerging roles of transcriptional programs in autophagy regulation.

Autophagy is an essential cellular process that degrades cytoplasmic organelles and components. Precise control of autophagic activity is achieved by context-dependent signaling pathways. Recent studies have highlighted the involvement of transcriptional programs during autophagic responses to various signals.

Transcriptional regulation of autophagy and lysosomal function by bromodomain protein BRD4.

Macroautophagy/autophagy is an intracellular recycling system that delivers cytoplasmic organelles and materials to lysosomes for degradation. This process is operated by autophagy-related (ATG) genes and tightly controlled by stress-responsive signaling pathways. Our recent study revealed that autophagy programs are transcriptionally suppressed by the BET family protein BRD4.

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis.

Molecular Pathways Controlling Autophagy in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the few cancer types where the 5-year survival rate shows no improvement. Despite conflicting evidence, the majority of data points to an essential role for autophagy in PDAC growth and survival, in particular constitutively activated autophagy, can provide crucial fuel to PDAC tumor cells in their nutrient-deprived environment. Autophagy, which is required for cell homeostasis, can both suppress and promote tumorigenesis and tumor survival in a context-dependent manner.

Non-transcriptional Function of FOXO1/DAF-16 Contributes to Translesion DNA Synthesis.

Forkhead box O (FOXO; DAF-16 in nematode) transcription factors activate a program of genes that control stress resistance, metabolism, and lifespan. Given the adverse impact of the stochastic DNA damage on organismal development and ageing, we examined the role of FOXO/DAF-16 in UV-induced DNA-damage response. Knockdown of FOXO1, but not FOXO3a, increases sensitivity to UV irradiation when exposed during S phase, suggesting a contribution of FOXO1 to translesion DNA synthesis (TLS), a replicative bypass of UV-induced DNA lesions.

Autophagy Determines the Path on the TRAIL to Death.

Macroautophagy facilitates degradation of cellular constituents and can positively or negatively affect cell death depending on the context. In this issue of Developmental Cell, Goodall and colleagues (2016) add to this complexity by showing that autophagy regulators can determine not only cell viability, but also the mechanism by which cells die.

Role of the SIK2-p35-PJA2 complex in pancreatic β-cell functional compensation.

Energy sensing by the AMP-activated protein kinase (AMPK) is of fundamental importance in cell biology. In the pancreatic β-cell, AMPK is a central regulator of insulin secretion. The capacity of the β-cell to increase insulin output is a critical compensatory mechanism in prediabetes, yet its molecular underpinnings are unclear.

GSK3β regulates gluconeogenic gene expression through HNF4α and FOXO1.

Hepatic gluconeogenesis is important for the maintenance of blood glucose homeostasis under fasting condition. Hepatocyte nuclear factor 4α (HNF4α) and FOXO1 transcription factors have implicated in this process through transcriptional regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which are rate-limiting enzymes in gluconeogenesis. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates the expression of gluconeogenic genes through HNF4α and FOXO1.

Arginine methylation of BCL-2 antagonist of cell death (BAD) counteracts its phosphorylation and inactivation by Akt.

Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins.

Regulation of FoxO transcription factors by acetylation and protein-protein interactions.

The forkhead box O transcription factors convert a variety of external stimuli, including growth factors, nutrients, and oxidative stress, into diverse biological responses through modulation of specific gene expression. Forkhead box O regulation is principally achieved by two distinct mechanisms: post-translational modifications and protein-protein interactions. Among several modifications of forkhead box O factors, we focus on reversible acetylation, describing past research and current advances.

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1.

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein.