Helene Knævelsrud

Fighting disease by selective autophagy of aggregate‐prone proteins

Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin‐proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel‐shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate‐prone proteins and the relevance of this process to protein aggregate associated diseases.

Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation

SNX18 promotes autophagosome formation by remodeling membranes and providing membrane to forming autophagosomes.

UVRAG mutations associated with microsatellite unstable colon cancer do not affect autophagy.

Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI ) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HE K cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.

Lipids in autophagy: Constituents, signaling molecules and cargo with relevance to disease ☆

The balance between protein and lipid biosynthesis and their eventual degradation is a critical component of cellular health. Autophagy, the catabolic process by which cytoplasmic material becomes degraded in lysosomes, can be induced by various physiological stimuli to maintain cellular homeostasis. Autophagy was for a long time considered a non-selective bulk process, but recent data have shown that unwanted components such as aberrant protein aggregates, dysfunctional organelles and invading pathogens can be selectively eliminated by autophagy. Recently, also intracellular lipid droplets were described as specific autophagic cargo, indicating that autophagy plays a role in lipid metabolism and storage (Singh et al., 2009 [1]). Moreover, over the past several years, it has become increasingly evident that lipids and lipid-modifying enzymes play important roles in the autophagy process itself, both at the level of regulation of autophagy and as membrane constituents required for formation of autophagic vesicles. In this review, we will discuss the interplay between lipids and autophagy, as well as the role of lipid-binding proteins in autophagy. We also comment on the possible implications of this mutual interaction in the context of disease. This article is part of a Special Issue entitled Lipids and Vesicular Transport.

SNX18 tubulates recycling endosomes for autophagosome biogenesis

The role of membrane remodeling and phosphoinositide-binding proteins in autophagy remains elusive. PX domain proteins bind phosphoinositides and participate in membrane remodeling and trafficking events and we therefore hypothesized that one or several PX domain proteins are involved in autophagy. Indeed, the PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation using an image-based siRNA screen. We show that SNX18 interacts with ATG16L1 and LC3, and functions downstream of ATG14 and the class III PtdIns3K complex in autophagosome formation. SNX18 facilitates recruitment of ATG16L1 to perinuclear recycling endosomes, and its overexpression leads to tubulation of ATG16L1- and LC3-positive membranes. We propose that SNX18 promotes LC3 lipidation and tubulation of recycling endosomes to provide membrane for phagophore expansion.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila.

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.

Apical Accumulation of the Sevenless Receptor Tyrosine Kinase During Drosophila Eye Development Is Promoted by the Small GTPase Rap1

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other corequired signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell–cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.

HS1BP3 inhibits autophagy by regulation of PLD1

ABSTRACT Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.