Nature Communications: Cholesterol transporters influence cancer development by regulating autophagy and mitochondria

Autophagy refers to the process
by which damaged organelles, long-lived proteins, pathogens and other cytoplasmic components are transported to lysosomes for degradation.
Depending on the way the degradation substrate is transported to the lysosome, autophagy can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy
.
What is commonly referred to as autophagy refers to macroautophagy
.
In the process of macroautophagy, a double membrane is first formed around the substrate to be degraded, autophagosomes are formed, and the endosomes or lysosomes are fused to form autolysosomes, and then under the action of V-ATPase proton pump on the membrane surface, the pH value in the cavity drops to activate the relevant hydrolase and degrade the
substrate.

As a key link in the initiation of autophagy, the formation of autophagosome membrane is of great significance
.
In the early stages of autophagy initiation, the autophagosome membrane lacks transmembrane proteins, and its regulation may mainly rely on membrane-associated proteins and the lipid components
of the membrane itself.
Previous studies have shown that autophagosome membranes lack cholesterol and that short-term removal of cholesterol from cells or long-term increase in cholesterol levels can increase autophagy levels
.
These findings suggest that cholesterol metabolism may be one of the
key factors affecting autophagy levels.

The GRAMD protein family is a class of proteins
that bind to phosphatidylserine on cell membranes via the characteristic GRAM domain.
Known GRAMD proteins include GRAMD1A, GRAMD1B, GRAMD1C, GRAMD2, GRAMD3, of which only GRAMD1A, GRAMD1B, GRAMD1C, these three proteins, can mediate the transport
of cholesterol from the cell membrane to the endoplasmic reticulum through their own sterol-binding domain VASt.
Studies have found that GRAMD1A is necessary for
autophagy to start.
Since GRAMD proteins often form heterologous complexes to work together, it is likely that other GRAMD proteins regulate autophagy levels
by regulating intracellular cholesterol metabolism.

Recently, Professor Anne Simonsen of the University of Oslo published a report in Nature Communications entitled The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics Research papers
.

This study found that whether drugs to reduce cell membrane cholesterol levels or interfere with the expression of the GRAMD1C protein, it was possible to increase the level
of autophagy in U2OS cells (human osteosarcoma cells).
In addition, knockdown/knockout of GRAMD1C protein increases mitochondrial cholesterol content and upregulates mitochondrial ATP-coupled aerobic respiration rate
.
Finally, the research team analyzed the TCGA database and revealed a relationship
between the GRAMD protein family and the prognosis of renal clear cell carcinoma.
This study enlightens us to think about the molecular mechanisms
behind autophagy and cancer prognosis from the perspective of cellular cholesterol metabolism.

When nutrients are deficient, cells often initiate autophagy, providing essential amino acids and other raw materials
for life activities.
Methyl β cyclodextrin (MBCD) quickly removes cholesterol from cell membrane components, while atorvastatin (ATV) removes cholesterol from cells for a long time
.
The research team induced autophagy through EBSS medium, and they found that after MBCD/ATV treatment, the autophagy level of cells increased, and the expression of autophagy initiation complex increased
.
In addition, they also found that MBCD/ATV treatment can cause autophagy to occur earlier without degradation of mTOR inhibition signal, and upregulate the expression level of autophagy membranes enriched with PtdIns(3) P enriched in the early bending of autophagy, suggesting that cholesterol elimination can affect the dynamic process
of autophagy.

After MBCD treatment, the level of autophagy increased and occurred earlier

On this basis, the research team used siRNA and CRISPR/Cas9 technology to study the role of
cholesterol transporters in cells in autophagy.
They found that knocking down/knocking out the GRAMD1C protein could upregulate the autophagy level of cells: increasing the number of autophagic lysosomes, increasing the radioactivity of the supernatant after 14C valine culture, increasing LC3-II autophagy flow, increasing the expression of autophagy initiation complexes, and upregulating the expression level
of curved autophagy membranes.

After knocking down the GRAMD1C protein, the level of autophagy increased, and the expression of autophagy initiation complex increased

In summary, the research team believes that the GRAMD1C protein can inhibit the biosynthesis
of autophagosomes by inhibiting the formation of curved autophagy membranes in the early stages of autophagy and the recruitment of autophagy initiation complexes.

Since previous studies have shown that autophagosome formation sites are often associated with ER-Mit contact sites, and the homologous sequence Lam6 of GRAM in yeast cells tends to be highly expressed at vesicle contact sites, the research team further explored whether GRAMD1C protein is expressed at ER-Mit contact sites and even affects mitochondrial function
.

Anne Simonsen's team found that the GRAMD1C protein was not only spatially co-expressed with ER and Mit, but also interacted with the mitochondrial outer membrane proteins TOMM70A and RHOT2
.
On this basis, the research team knocked down/knocked out the GRAMD1C protein and found that without affecting mitochondrial morphology, mitochondrial proteome, mitochondrial membrane potential and cellular ROS level, the cholesterol content of mitochondria increased, the cholesterol content of the endoplasmic reticulum decreased, and the aerobic respiration rate of mitochondrial ATP-coupled increased
.
It is suggested that GRAMD1C protein can mediate cholesterol transport between the endoplasmic reticulum and mitochondria and inhibit mitochondrial bioenergy
.

Knockdown/knockout of GRAMD1C protein, increased mitochondrial cholesterol content, and increased aerobic respiration rate

Finally, the research team used the disease model of clear cell carcinoma of kidney and analyzed the TCGA database to reveal that GRAMD1C and GRAMD2B are unfavorable factors in the prognosis of kidney cancer, and GRAMD1A and GRAMD1B are favorable factors for the prognosis of
kidney cancer.

Professor Anne Simonsen said that the study found that down-regulation of cell membrane cholesterol is beneficial to the recruitment of autophagy initiation proteins and promotes the synthesis of autophagosomes; GRAMD1C protein may inhibit autophagosome synthesis and downregulate mitochondrial bioenergy
by regulating cholesterol transport between cell membranes and endoplasmic reticulum, and between endoplasmic reticulum and mitochondria.
These new findings are also related to the colony, proliferation, migration and prognosis of cancer cells, and the next step should focus on the role
of cholesterol regulation in the biofilm system in autophagy and cancer progression.

Original source:

Ng， M.
Y.
W.
， Charsou， C.
， Lapao， A.
et al.
The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics.
Nat Commun 13， 6283 (2022).
https：//doi.
org/10.
1038/s41467-022-33933-2.