Efficient Transport Of The Accessible Pool Of Pm Cholesterol To The Er Requires Gramd1 Complex Formation
A version of GRAMD1b in which the transmembrane domain and luminal region are both replaced by those of Sec61Î² cannot form protein complexes . Remarkably, GRAMD1b TM swap failed to rescue the reduced suppression of SREBP-2 cleavage observed in GRAMD1 TKO cells and failed to suppress the enhanced recruitment of EGFPâGRAM1b to the PM in TKO cells upon sphingomyelinase treatment, although the mutant protein was still recruited to the PM . TIRF microscopy analysis of HeLa cells expressing the GRAMD1b TM swap mutant, however, revealed major differences in how this protein was recruited to the PM compared to wild-type GRAMD1b . GRAMD1b TM swap remained diffusely distributed on the tubular ER even at the end of the 180 min imaging period. By contrast, wild-type GRAMD1b progressively accumulated at ERâPM contacts as discrete patches with much stronger PM recruitment . These results support an important role for GRAMD1 complex formation in facilitating the progressive accumulation of GRAMD1s at ERâPM contacts, thereby supporting efficient accessible cholesterol transport at these contacts. Taken together, we conclude that GRAMD1s play a role in PM to ER transport of the accessible pool of PM cholesterol upon acute expansion of this pool. Loss of GRAMD1 function leads to sustained accumulation of accessible cholesterol in the PM, resulting in less effective suppression of SREBP-2 cleavage and possibly dysregulation of cellular cholesterol homeostasis.
Cholesterol’s Effects On Cellular Membranes
- Virginia Tech
- New findings have far-reaching implications in the general understanding of disease, the design of drug delivery methods, and many other biological applications that require specific assumptions about the role of cholesterol in cell membranes.
For more than a decade, scientists have accepted that cholesterol — a key component of cell membranes — did not uniformly affect membranes of different types. But a new study led by Assistant Professor Rana Ashkar of the Virginia Tech Department of Physics finds that cholesterol actually does adhere to biophysical principles.
The findings, published recently in the Proceedings of the National Academy of Sciences, have far-reaching implications in the general understanding of disease, the design of drug delivery methods, and many other biological applications that require specific assumptions about the role of cholesterol in cell membranes.
“Cholesterol is known to promote tighter molecular packing in cell membranes, but reports about how it stiffens membranes have been so conflicting,” said Ashkar, who is a faculty member in the Virginia Tech College of Science. “In this work, we show that, at the nanoscale level, cholesterol indeed causes membrane stiffening, as predicted by physical laws. These findings affect our understanding of the biological function of cholesterol and its role in health and disease.”
Cholesterol’s impact on cell membranes at the molecular level
Proving her point
How Does Cholesterol Affect The Cell Membrane
At high temperatures, cholesterol interferes with the movement of the phospholipid fatty acid chains, making the outer part of the membrane less fluid and reducing its permeability to small molecules. Although cholesterol is not present in bacteria, it is an essential component of animal cell plasma membranes.
What is the function of cholesterol in the phospholipid bilayer?
Biological membranes typically include several types of molecules other than phospholipids. A particularly important example in animal cells is cholesterol, which helps strengthen the bilayer and decrease its permeability. Cholesterol also helps regulate the activity of certain integral membrane proteins.
The Cholesterol Transporting Property Of The Start
The cholesterol transporting property of the StART-like domain of GRAMD1s is critical for removal of an acutely expanded pool of accessible PM cholesterol.
Figure 5âsource data 1
Guided by the crystal structures of GRAMD1 StART-like domains in complex with 25-hydroxycholesterol , we designed mutations that would potentially block the insertion of cholesterol into the GRAMD1b StART-like domain. Our mutagenesis strategy was to rigidify the loop that was predicted to open or close to capture or release sterol . Purified GRAMD1a and GRAMD1b StART-like domains with 5P mutations were unable to transfer DHE in vitro . A similar result was also obtained with a version of the GRAMD1b StART-like domain with a point mutation that was previously shown to be defective in DHE extraction in vitro .
Taken together, our results suggest a critical role of the GRAMD1s in controlling the movement of the accessible pool of PM cholesterol between the PM and the ER via their StART-like domains.
Cholesterol Functions In The Cell Membrane
The cholesterol in the cell membrane achieve the following functions
- Structure of the cell and membrane
It is due to the presence of cholesterol molecules that cells get their structure. Cells with well-defined cell membranes exhibit distinct existence from surrounding cells. The presence of HDL in cell membranes accords them the required transmission capabilities to achieve balanced cell nutrition.
- Conduct of intercellular functions
An efficient cell membrane allows for the efficient conduct of intercellular processes with the cells. Within the cell, the cell organelles release chemicals and absorb molecules to synthesize and break down substances. A cell membrane of appropriate structure maintains boundaries and does not rupture untimely.
- Reverse transfer vehicles
HDL from cell membrane serves as vehicles for the reverse transfer of LDL to the liver where they get converted to bile. Thus HDL helps maintain the correct cholesterol balance and reduce excess LDL in the body.
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Gramd Proteins Form Homo
Previous studies identified GRAMD1s as ER-resident proteins that are distributed throughout ER structures in a punctate pattern . GRAMDs all possess an N-terminal GRAM domain and a C-terminal transmembrane domain. In addition, the three GRAMD1 proteins possess a StART-like domain . Some LTPs are known to form homo- and heteromeric complexes. Thus, we reasoned that GRAMD1s may also interact with one another to form complexes. To further analyze the dynamics of these proteins on the ER at high spatial resolution, we tagged the GRAMD1s, as well as GRAMD3, with fluorescent proteins and analyzed their localization using spinning disc confocal microscopy coupled with structured illumination . Analysis of COS-7 cells expressing individual EGFP-tagged GRAMD1s or GRAMD3 and a general ER marker revealed enrichment of GRAMD1s and GRAMD3 in similar discrete patches along ER tubules. By contrast, RFP-Sec61Î² localized to all domains of the ER, including the nuclear envelope and the peripheral tubular ER network . When individual EGFPâGRAMD1s and either mRuby-tagged GRAMD1b or mCherry-tagged GRAMD3 were co-expressed in COS-7 cells, the patches of EGFP and mRuby/mCherry significantly overlapped, indicating potential complex formation between these proteins on tubular ER.
GRAMD proteins form homo- and heteromeric complexes.
Cholesterol Structure Dynamics And Membrane Topology
Cholesterol is a polycyclic amphipathic molecule derived from the sterane backbone . Its polar section is restricted to a single hydroxyl group which can form two distinct types of hydrogen bond with a polar group belonging to either a membrane lipid or a protein. The apolar section of cholesterol has an asymmetric structure with two distinct faces, referred to as and according to the system numeration of ring compounds proposed by Rose et al. . The face displays a planar surface, in contrast with the face which has a significantly rougher surface owing to the presence of several aliphatic groups . The side chains of branched amino acids such as Ile, Val, or Leu can interpenetrate these aliphatic spikes and are thus particularly suited for an association with the face of cholesterol through van der Waals interactions. This is the case for the cholesterol binding domain of -synuclein . Moreover, aromatic side chains can stack onto the face of cholesterol through CH- interactions . However, this should not be taken as an absolute rule since the aliphatic side chains of an -helical segment could also form a groove with a planar surface fitting the face of cholesterol . Conversely, an aromatic ring oriented normally with respect to the main axis of an -helical region could perfectly well accommodate the rough face of cholesterol by intercalating the aromatic structure between the aliphatic spikes of the lipid.
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How Is High Cholesterol Diagnosed
There are usually no signs or symptoms that you have high cholesterol. There is a blood test to measure your cholesterol level. When and how often you should get this test depends on your age, risk factors, and family history. The general recommendations are:
For people who are age 19 or younger:
- The first test should be between ages 9 to 11
- Children should have the test again every 5 years
- Some children may have this test starting at age 2 if there is a family history of high blood cholesterol, heart attack, or stroke
For people who are age 20 or older:
- Younger adults should have the test every 5 years
- Men ages 45 to 65 and women ages 55 to 65 should have it every 1 to 2 years
What Can Raise My Risk Of High Cholesterol
A variety of things can raise your risk for high cholesterol:
- Age. Your cholesterol levels tend to rise as you get older. Even though it is less common, younger people, including children and teens, can also have high cholesterol.
- Heredity. High blood cholesterol can run in families.
- Weight. Being overweight or having obesity raises your cholesterol level.
- Race. Certain races may have an increased risk of high cholesterol. For example, African Americans typically have higher HDL and LDL cholesterol levels than whites.
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The Gram Domain Of Gramd1s Acts As A Coincidence Detector Of Unsequestered/accessible Cholesterol And Anionic Lipids And Senses The Accessibility Of Cholesterol
Recent studies demonstrated that âcholesterol loadingâ leads to the accumulation of GRAMD1s at ERâPM contact sites . Within 20 min of treating cells with a complex of cholesterol and methyl-Î²-cyclodextrin , GRAMD1b was indeed recruited to the PM . In addition, we found that GRAMD1a, GRAMD1c, and GRAMD3 were all recruited to ERâPM contacts upon cholesterol loading, with kinetics similar to GRAMD1b recruitment . However, a version of GRAMD1b that lacked the GRAM domain failed to localize to the PM, even after 30 min, indicating the essential role of this domain in sensing PM cholesterol . Although these results suggest that PM cholesterol plays a critical role in recruiting GRAMDs to ERâPM contacts, all of the GRAMDs localize to tubular ER at rest, even though a significant amount of cholesterol is already present in the PM . Thus, their GRAM domains may possess unique abilities to sense the accessibility of PM cholesterol, rather than detecting the total levels of PM cholesterol. However, it is not known whether the GRAM domains are able to sense accessible cholesterol in the PM.
The GRAM domain of GRAMD1s acts as a coincidence detector of unsequestered/accessible cholesterol and anionic lipids, and senses a transient expansion of the accessible pool of cholesterol in the PM.
Figure 3âsource data 1
GRAMD1b is recruited to ERâPM contacts upon cholesterol loading.
What Causes High Cholesterol
The most common cause of high cholesterol is an unhealthy lifestyle. This can include
- Unhealthy eating habits, such as eating lots of bad fats. One type, saturated fat, is found in some meats, dairy products, chocolate, baked goods, and deep-fried and processed foods. Another type, trans fat, is in some fried and processed foods. Eating these fats can raise your LDL cholesterol.
- Lack of physical activity, with lots of sitting and little exercise. This lowers your HDL cholesterol.
- Smoking, which lowers HDL cholesterol, especially in women. It also raises your LDL cholesterol.
Genetics may also cause people to have high cholesterol. For example, familial hypercholesterolemia is an inherited form of high cholesterol. Other medical conditions and certain medicines may also cause high cholesterol.
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Where Are Lipids Found In The Cell Membrane
4.1/5cell membranelipidcell membranes
The three major classes of membrane lipids are phospholipids, glycolipids, and cholesterol.
Similarly, which lipid is not found in cell membranes? The lipid bilayer of many cell membranes is not composed exclusively of phospholipids, however it often also contains cholesterol and glycolipids. Eucaryotic plasma membranes contain especially large amounts of cholesterol up to one molecule for every phospholipid molecule.
Moreover, where are lipids found in the cell?
They can be found in many parts of a human: cell membranes, cholesterol, blood cells, and in the brain, to name a few ways the body uses them. Lipids are important for cell membrane structure, regulating metabolism and reproduction, the stress response, brain function, and nutrition.
How do lipids make up the cell membrane?
Phospholipids make up the basic structure of a cell membrane. This arrangement of phospholipid molecules makes up the lipid bilayer. The phospholipids of a cell membrane are arranged in a double layer called the lipid bilayer. The hydrophilic phosphate heads are always arranged so that they are near water.
What Are Cellular Membranes Made Of
With few exceptions, cellular membranes including plasma membranes and internal membranes are made of glycerophospholipids, molecules composed of glycerol, a phosphate group, and two fatty acid chains. Glycerol is a three-carbon molecule that functions as the backbone of these membrane lipids. Within an individual glycerophospholipid, fatty acids are attached to the first and second carbons, and the phosphate group is attached to the third carbon of the glycerol backbone. Variable head groups are attached to the phosphate. Space-filling models of these molecules reveal their cylindrical shape, a geometry that allows glycerophospholipids to align side-by-side to form broad sheets .
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Deletion Of Gramd1s Results In Exaggerated Accumulation Of The Accessible Pool Of Cholesterol In The Pm
As GRAMD1s move to ERâPM contact sites upon acute expansion of the accessible pool of PM cholesterol , they may also contribute to the extraction of accessible PM cholesterol in order to maintain homeostasis. To investigate the potential functions of GRAMD1s in this process, we used the CRISPR/Cas9 system to disrupt GRAMD1 function by targeting all three GRAMD1 genes in HeLa cells. Guide RNAs specific to exon 13 of GRAMD1A and GRAMD1B and to exon 11 of GRAMD1C were chosen, as they encode the lipid-harboring StART-like domains . After transfection of plasmids expressing GRAMD1-specific guide RNAs and Cas9 protein, two independent isolates of GRAMD1a/1b double knockout cell clones and two independent isolates of GRAMD1a/1b/1c triple knockout cell clones were selected. The absence of GRAMD1a and GRAMD1b was confirmed by western blotting and genomic sequencing . Disruption of the GRAMD1C gene was validated by sequencing the targeted genomic region within the GRAMD1C locus . No obvious defects in cell viability or overall morphology were observed for these KO cells, with the exception that KO cells grew slightly slower than parental HeLa cells. Subsequent experiments were performed using GRAMD1a/1b/1c TKO #15 cells .
Deletion of GRAMD1s results in exaggerated accumulation of the accessible pool of cholesterol in the PM.
Figure 4âsource data 1
Gramd1s Play A Role In Accessible Cholesterol Transport From The Pm To The Er During Acute Expansion Of The Accessible Pool Of Pm Cholesterol
Acute expansion of the accessible pool of PM cholesterol results in the suppression of SREBP-2 cleavage and the inhibition of cholesterol biosynthesis as a result of transport of accessible cholesterol from the PM to the ER. However, the intracellular transport machinery by which accessible cholesterol is transported from the PM to the ER remains unknown. GRAMD1s may play a role in this process, as they are able to sense and counteract the acute expansion of the accessible pool of PM cholesterol.
GRAMD1s-mediated PM to ER cholesterol transport plays a role in the suppression of SREBP-2 cleavage upon sphingomyelinase treatment.
Figure 6âsource data 1
Comparison of the recruitment to the PM of a wild-type GRAMD1b and of a mutant version of GRAMD1b that is defective in complex formation upon sphingomyelinase treatment.
HeLa cells expressing EGFPâGRAMD1b or EGFPâGRAMD1b TM swap were imaged under TIRF microscopy. Images were taken every 20 s, and 100 mU/ml of sphingomyelinase was added at the 10 min time point. Image size, 66.1 Âµm x 66.1 Âµm.
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What Health Problems Can High Cholesterol Cause
If you have large deposits of plaque in your arteries, an area of plaque can rupture . This can cause a blood clot to form on the surface of the plaque. If the clot becomes large enough, it can mostly or completely block blood flow in a coronary artery.
Plaque also can build up in other arteries in your body, including the arteries that bring oxygen-rich blood to your brain and limbs. This can lead to problems such as carotid artery disease, stroke, and peripheral arterial disease.
Effects Of Membrane Cholesterol On Cell Secretion
Model of exocytosis triggered by cholesterol sequestration from plasma membrane. Plasma membrane containing cholesterol and presence formation of the membrane rafts microdomains. In these domains, actin-binding proteins hold actin filaments in specific locations. Peripheral lysosomes are docked to these actin filaments. Plasma membrane after cholesterol sequestration. Actin-binding proteins reallocate along the membrane due to the disruption of the membrane rafts. Actin filaments also increase in size due to actin polymerization induced by cholesterol sequestration. In this scenario, reorganized actin filaments push peripheral lysosomes closer to the cell plasma membrane secretion occurs.
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