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. Several aspects of the role of specific lipids in autophagy are poorly understood, such as the lipid composition of the autophagosomal membrane and the involvement of lipid-binding proteins and lipid-modifying enzymes in altering the properties of forming autophagosomes. To approach some of these questions we have focused on PX domain-containing proteins, which are known to bind phosphoinositides (PIs), but little is known about their function in autophagy.To search for PX proteins in autophagy, we performed an imaging-based siRNA screen, where HS1BP3 was identified as a putative negative regulator of autophagy. Knockdown of HS1BP3 increases LC3 puncta formation and LC3 lipidation, both under basal and starved conditions. Autophagic flux assays indicate that HS1BP3 functions during autophagosome formation. Accordingly, depletion of HS1BP3 increases the degradation of the autophagic cargo SQSTM1/p62 and longlived proteins. For in vivo analysis, HS1BP3 was silenced in transgenic GFP-LC3 zebrafish and as observed in cells, the number of GFP-LC3 puncta increases in embryos lacking HS1BP3. This increase was partly rescued by co-injecting human HS1BP3-mRNA, indicating that HS1BP3 negatively regulates autophagy also in zebrafish.HS1BP3 is generally cytosolic, but also enriched on structures containing autophagy proteins ATG9 and ATG16L1 as well as recycling endosome markers TF (transferrin) and TFRC (transferrin receptor) (Fig. 1). Live-cell imaging revealed that these structures fuse with LC3-positive vesicles, indicating that they represent recycling endosome-derived membranes providing input to forming autophagosomes. This is in line with recent findings showing that ATG9-and ATG16L1-positive vesicles traffic via the plasma membrane through recycling endosomes and that such trafficking is important for a...

MiR‐122‐5p and miR‐326‐3p promote cadmium‐induced NRK‐52E cell apoptosis by downregulating PLD1

“…PLD1 has been shown to promote cell survival and protect against apoptosis via mediating several signaling pathways 14 . In particular, PLD1 can catalyze phosphatidylcholine (PC) to produce phosphatidic acid (PA), which plays an essential role in autophagy 23 . Accumulative evidence has shown that autophagy is indispensable for kidney proximal tubular cell survival, 24‐26 so the cadmium‐induced cell death and kidney injury in this study may be attributed, at least partially, to the impaired autophagy resulted from PLD1 downregulation, but further experiment is needed to testify it.…”

Phospholipase D1 Attenuation Therapeutics Promotes Resilience against Synaptotoxicity in 12-Month-Old 3xTg-AD Mouse Model of Progressive Neurodegeneration

“…More elaborate studies using proximity ligation assay will be required to quantify further the importance and relevance of this association from a therapeutic point of view. Since PLD1 levels were elevated in the astrocytic and mitochondrial compartments in the previously reported study, our future studies will explore mechanisms in which PLD1 has been implicated and plays an important role in the progression of neurodegenerative states including autophagy [ 29 , 94 , 95 , 96 ], neuroinflammation [ 35 , 64 , 97 , 98 , 99 , 100 , 101 , 102 ], reactive oxygen species [ 103 , 104 , 105 , 106 ] and infection [ 35 , 107 , 108 , 109 ] and how these contribute to the synaptic dysfunction leading to cognitive decline in our studies.…”

Phospholipase D1 promotes cervical cancer progression by activating the RAS pathway

“…PLD1 can hydrolyse major membrane glycerophospholipids to the lipid second messenger PA, which plays a role in disease processes such as cancer 11 . PLD1 and its product, PA, participate in various physiological and pathological processes, such as cell growth, invasion, metabolism and autophagy 10,12,13 . PLD1 exhibits a higher expression level in colorectal tumours, bladder cancer and breast cancer 14–16 .…”

“…11 PLD1 and its product, PA, participate in various physiological and pathological processes, such as cell growth, invasion, metabolism and autophagy. 10,12,13 PLD1 exhibits a higher expression level in colorectal tumours, bladder cancer and breast cancer. [14][15][16] The high expression of PLD1 in tumour tissues is related to a poor prognosis in patients with colorectal cancer and bladder cancer.…”