FLCN and Autophagy
1. Basic about autophagy:
Autophagy (or autophagocytosis) is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery. The breakdown of cellular components can ensure cellular survival during starvation by maintaining cellular energy levels. Autophagy, if regulated, ensures the synthesis, degradation and recycling of cellular components. During this process, targeted cytoplasmic constituents are isolated from the rest of the cell within the autophagosomes, which are then fused with lysosomes and degraded or recycled. There are three different forms of autophagy that are commonly described, which include macroautophagy, microautophagy and chaperone-mediated autophagy. In the context of disease, autophagy has been seen as an adaptive response to survival, whereas in other cases it appears to promote cell death and morbidity.
There are three main pathways involved in autophagy and these are mediated by the autophagy-related genes and their associated proteases.
Macroautophagy is the main pathway, occurring mainly to eradicate damaged cell organelles or unused proteins. This involves the formation of a double membrane around the organelle known as an autophagosome. This formation is induced by class 3 phosphoinositide-3-kinase, the autophagy-related gene (Atg) 6 (also known as Beclin-1) and ubiquitin complexes. Other proteases such as Atg4, Atg12, Atg5, and Atg16 are also involved in the regulation of these pathways. The autophagosome travels through the cytoplasm of the cell to a lysosome, and the two membranes fuse together allowing the autophagosome to enter via endocytosis.Within the lysosome, the contents of the autophagosome are degraded via acidic lysosomal hydrolases.
Microautophagy, on the other hand, involves the direct engulfment of cytoplasmic material into the lysosome. This occurs by invagination, meaning the inward folding of the lysosomal membrane, or cellular protrusion.
Chaperone-mediated autophagy, or CMA, is a very complex and specific pathway, which involves the recognition by the hsc70-containing complex. This means that a protein must contain the recognition site for this hsc70 complex which will allow it to bind to this chaperone, forming the CMA- substrate/chaperone complex. This complex then moves to the lysosomal membrane-bound protein that will recognise and bind with the CMA receptor, allowing it to enter the cell. Upon recognition, the substrate protein gets unfolded and it is translocated across the lysosome membrane with the assistance of the lysosomal hsc70 chaperone. CMA is significantly different from other types of autophagy because it translocates protein material in a one by one manner, and it is extremely selective about what material crosses the lysosomal barrier.
Functions:
1). Nutrient starvation
Autophagy has important roles in various cellular functions, one particular example is in yeasts, where the nutrient starvation induces a high level of autophagy, which allows unneeded proteins to be degraded and the amino acids recycled for the synthesis of proteins that are essential for survival. In higher eukaryotes, autophagy is also induced in response to the nutrient depletion that occurs in animals at birth after severing of the trans-placental food supply, as well as that of nutrient starved cultured cells and tissues. Mutant yeast cells that have a reduced autophagic capability rapidly perish in nutrition-deficient conditions. Studies on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast. A gene known as Atg7 has been implicated in nutrient-mediated autophagy, as mice studies have shown that starvation-induced autophagy was impaired in Atg7-deficient mice.
2). Infection
Autophagy has been recognized as an immune mechanism. It plays a role in the destruction of intracellular pathogens, in a process of degradation of dysfunctional intracellular organelles. Intracellular pathogens such as Mycobacterium tuberculosis is the bacterium which is responsible for tuberculosis and it can survive within the cells by blocking the maturation of its phagosome into a degradative organelle called a phagolysosome. Stimulation of autophagy in infected cells helps overcome the block and aids the cell to eliminate the pathogens. The virus (Vesicular stomatitis virus) is believed to be taken up by the autophagosome from the cytosol and translocated to the endosomes where detection takes place by a member of the PRRs called toll-like receptor 7, detecting single stranded RNA. Following activation of the toll-like receptor, intracellular signaling cascades are initiated, leading to induction of interferon and other other antiviral cytokines. A subset of viruses and bacteria subvert the autophagic pathway to promote their own replication. Galectin-8 has recently been identified as an intracellular "danger receptor", able to initiate autophagy against intracellular pathogens. When galectin-8 binds to a damaged vacuole, it recruits autophagy adaptor such as NDP52 leading to the formation of an autophagosome and bacterial degradation.
3). Repair mechanism
Autophagy degrades damaged organelles, cell membranes and proteins, and the failure of autophagy is thought to be one of the main reasons for the accumulation of cell damage and aging.
4). Programmed cell death
Apoptosis (cell suicide) is not the only form of programmed cell death (PCD). Recent studies have provided evidence that there is another mechanism of PCD which is associated with the appearance of autophagosomes and depends on autophagy proteins. This form of cell death most likely corresponds to a process that has been morphologically defined as autophagic PCD. This hypothesis as well as previous reports that cells with autophagic features often exist in regions where PCD is occurring seem to support the existence of autophagic PCD. One question that constantly arises, however, is whether autophagic activity in dying cells is the cause of death or is actually an attempt to prevent it. Morphological and histochemical studies cannot prove a causative relationship between the autophagic process and cell death. In fact, there have recently been strong arguments that autophagic activity in dying cells might actually be a survival mechanism. Studies of the metamorphosis of insects have shown cells undergoing a form of PCD that appears distinct from other forms; these have been proposed as examples of autophagic cell death.
Macroautophagy (autophagy) in mammalian cells is a complex bulk degradation system involving autophagosome formation, maturation, and lysosomal fusion. Autophagy is controlled and co-ordinated by Atg signaling modules; the "ubiquitin-like protein (UBL) - Atg8" module is essential for autophagosome formation.
1). LC3 Protein and Autophagy
The mammalian homolog of Atg8 is called microtubule-associated proteins 1A/1B light chains 3A/LC3A and 3B/LC3B (MAP1LC3A/B), abbreviated to LC3. These ubiquitin-like proteins can be used as reliable autophagosome markers for monitoring autophagy.
LC3 is synthesized as pro-LC3 which is immediately processed by Atg4 into a cytosolic form, LC3-I. During autophagosome formation, LC3-I covalently links to phosphatidylethanolamine (PE)and is incorporated into autophagosome membranes where it recruits cargo. This event is mediated by Atg3 and Atg7. This lipidation process converts cytosolic LC3-I into the active, autophagosome membrane-bound form, LC3-II. It was recently shown that WIPI2, the human orthologue of Atg18, appears to be required for LC3 lipidation and is involved in positively regulating this process.
2). Upstream Events
Cellular stress conditions initiate upstream events through the MAPK/Erk1/2, PI3K/AKT and mammalian target of rapamycin (mTOR) pathways with mTORC complexes leading to the autophagy mechanisms macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) in eukaryotic cells.
Macroautophagy, often referred to as autophagy, is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic contents, abnormal protein aggregates, and excess or damaged organelles. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with physiological as well as pathological processes such as development, differentiation, neurodegenerative diseases, stress, infection and cancer. The kinase mTOR is a critical regulator of autophagy induction, with activated mTOR (Akt and MAPK signaling) suppressing autophagy, and negative regulation of mTOR (AMPK and p53 signaling) promoting it. Three related serine/threonine kinases, UNC-51-like kinase -1, -2, and -3 (ULK1, ULK2, UKL3), which play a similar role as the yeast Atg1, act downstream of the mTOR complex. ULK1 and ULK2 form a large complex with the mammalian homolog of an autophagy-related (Atg) gene product (mAtg13) and the scaffold protein FIP200 (an ortholog of yeast Atg17). Class III PI3K complex, containing hVps34, Beclin 1 (a mammalian homolog of yeast Atg6), p150 (a mammalian homolog of yeast Vps15), and Atg14-like protein (Atg14L or Barkor) or ultraviolet irradiation resistance-associated gene (UVRAG), is required for the induction of autophagy. The Atg genes control the autophagosome formation through Atg12-Atg5 and LC3-II (Atg8-II) complexes. Atg12 is conjugated to Atg5 in a ubiquitin-like reaction that requires Atg7 and Atg10 (E1 and E2-like enzymes, respectively). The Atg12–Atg5 conjugate then interacts noncovalently with Atg16 to form a large complex. LC3/Atg8 is cleaved at its C terminus by Atg4 protease to generate the cytosolic LC3-I. LC3-I is conjugated to phosphatidylethanolamine (PE) also in a ubiquitin-like reaction that requires Atg7 and Atg3 (E1 and E2-like enzymes, respectively). The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. Autophagy and apoptosis are connected both positively and negatively, and extensive crosstalk exists between the two. During nutrient deficiency, autophagy functions as a pro-survival mechanism; however, excessive autophagy may lead to cell death, a process morphologically distinct from apoptosis. Several pro-apoptotic signals, such as TNF, TRAIL, and FADD, also induce autophagy. Additionally, Bcl-2 inhibits Beclin-1-dependent autophagy, thereby functioning both as a pro-survival and as an anti-autophagic regulator.
3). Macroautophagy components
Macroautophagy is the most studied pathway involving the cellular degradation of cytoplasmic proteins, bulk cytoplasm, dysfunctional organelles and intracellular pathogens (innate immunity / cell defense mechanism). This process involves the formation of a phagophore which develops into a transport vesicle called the autophagosome containing the cytoplasmic constituents or organelles.
The autophagy machinery, including the phagophore assembly site / pre-autophagosomal structure (PAS), and its regulation involves more than 30 autophagy related genes (ATG Proteins) including Beclin 1, Activating molecule in beclin-1-regulated autophagy (AMBRA1), Damage-regulated autophagy modulator (DRAM), lysosome-associated membrane proteins (Lamp1 & 2 (CD107a & b) and Microtubule-associated proteins 1A/1B light chains 3A/B (MAP1LC3A/B).
4). Lysosome Detection – Cathepsin Antibodies and Detection Kits
The autophagosome fuses to a lysosome forming an autolysosome where enzymes such as cathepsins and permeases hydrolyze and degrade the cargo for its release back into the cytoplasm for recycling.
The Magic Red™ Cathepsin B, K and L Detection Kits are suitable for detection of lysosomes:
- Active cathepsin analysis in whole, living cells
- Substrate based assay
- Fluorescence microscopy or plate reader
A range of Cathepsin Antibodies are also available, and further details about our Cathepsin Detection Kits and antibodies are available here.
Future detailed understanding of autophagy mechanisms and their role in human disease and innate and adaptive immunity is needed before potential therapeutic intervention can be developed.
2. Autophagy Detection Methods
Autophagy can be measured by changes in LC3 localization: tracking the level of conversion of LC3-I to LC3-II provides an indicator of autophagic activity. In particular the levels of LC3-II correlate with autophagosome formation, due to its association with the autophagosome membrane.
A popular method for detecting autophagy uses antibodies specific to LC3 to identify LC3-positive structures such as autophagosomes. Since LC3 is the only protein identified on the inner and outer membranes of autophagosomes, MAP1LC3A/B antibodies provide a quick detection method.
In Western blotting, LC3 is detected as two bands; cytosolic LC3-I and LC3-II which is bound to PE in the autophagosome membrane. This makes the molecular weight of LC3-II greater than LC3-I. However due to its hydrophobicity, LC3-II migrates faster in SDS-PAGE and therefore displays a lower apparent molecular weight.
The MAP1LC3A/B (LC3A/B) polyclonal antibody recognizes both LC3-I and LC3-II forms. The antibody is superior for Western blotting, detecting two bands at 16-18 kDa (LC3-I) and 14-16 kDa (LC3-II).
This antibody is also validated for immunofluorescence. In autophagy the staining pattern has been shown to change from diffuse to more punctate when LC3-II is bound to the autophagosome (3).
The MAP1LC3A/B antibody is available in convenient and cost-effective pack sizes; Product Code AHP2167 0.1 mg and AHP2167T, 25 ug.
GFP-LC3 expression vectors are often used to transfect mammalian cells to study autophagy. We have GFP antibodies that can be used in a variety of applications to detect GFP fusion proteins.
