Chaperone-mediated autophagy (CMA) refers to the chaperone-dependent selection of soluble cytosolic proteins that are then targeted to lysosomes and directly translocated across the lysosome membrane for degradation. [1] The unique features of this type of autophagy are the selectivity on the proteins that are degraded by this pathway and the direct shuttling of these proteins across the lysosomal membrane without the requirement for the formation of additional vesicles (Figure 1).
The proteins that are degraded through CMA are cytosolic proteins or proteins from other compartments once they reach the cytosol. Therefore, some of the components that participate in CMA are present in the cytosol while others are located at the lysosomal membrane (Table I).
Specific selection of proteins for degradation in all forms of autophagy came to further understanding as studies discovered the role of chaperones like hsc70. Although hsc70 targets cytosolic protein to CMA based on specific amino acid sequence recognition, it works differently when targeting proteins to macro or microautophagy. [2]
In one mechanism for a protein to be a CMA substrate, it must have in its amino acid sequence a pentapeptide motif biochemically related to KFERQ. [3] This CMA-targeting motif is recognized by a cytosolic chaperone, heat shock cognate protein of 70 kDa (hsc70) which targets the substrate to the lysosome surface. [4] This substrate protein-chaperone complex binds to lysosome-associated membrane protein type 2A (LAMP-2A), which acts as the receptor for this pathway. [5] LAMP-2A a single span membrane protein, is one of the three spliced variants of a single gene lamp2. [6] The other two isoforms LAMP-2B and LAMP-2C are involved in macroautophagy and vesicular trafficking, respectively. Substrate proteins undergo unfolding after binding to LAMP-2A in a process likely mediated by the membrane associated hsc70 and its co-chaperones Bag1, hip, hop and hsp40, also detected at the lysosomal membrane. [7] This binding of substrates to monomers of LAMP-2A triggers the assembly of LAMP-2A multimers that act as the active translocation complex through which the substrates can pass through after unfolding. [8] Here, the translocation complex chooses only the substrate proteins which can unfold for internalization by the lysosomes. For instance, research with artificial CMA substrate showed that hsc70 chaperone binding to substrate or lysosomal binding does not necessarily require the substrate protein to be capable of unfolding, however, lysosomal translocation makes unfolding as a necessary criteria for it to be internalized. [2] Substrate translocation requires the presence of hsc70 inside the lysosomal lumen, which may act by either pulling substrates into the lysosomes or preventing their return to the cytosol. [9] After translocation the substrate proteins are rapidly degraded by the lysosomal proteases. Figure 1 depicts the different steps of CMA.
The limiting step for CMA is the binding of the substrate proteins to LAMP-2A and, consequently, levels of LAMP-2A at the lysosomal membrane correlate directly with CMA activity. Therefore, to modulate the activity of this autophagic pathway, the cell stringently regulates the levels of the CMA receptor at the lysosomal membrane by controlling the degradation rates of LAMP-2A monomers in lysosomes and by de novo synthesis of LAMP-2A molecules. In addition, transport of substrates also depends on the efficiency of the assembly of LAMP-2A into the translocation complex. [8]
Assembly and disassembly of CMA translocation complex is mediated by hsp90 and hsc70 chaperones, respectively. [8] Degradation of LAMP-2A monomers at the lysosomal membrane occurs in discrete cholesterol-rich lipid microdomains of the lysosomal membrane and it is mediated by Cathepsin A and an unidentified lysosomal metalloprotease. [10] Therefore, assembly, disassembly of LAMP-2A into active translocation complex, and its degradation in microdomain regions, highlights the dynamic nature of this process and the importance of lateral mobility of the CMA receptor at the lysosomal membrane.
CMA contributes to the maintenance of cellular homeostasis by facilitating recycling of amino acids of the degraded proteins (contribution to energetic cellular balance) and by eliminating abnormal or damaged proteins (contribution to cellular quality control). [11]
CMA is active at all times in different tissues (liver, kidney, brain), and almost all cell types in culture studied. However, it is maximally activated in response to stressors and changes in the cellular nutritional status. When nutrient supply is limited, the cells respond by activating autophagy, in order to degrade intracellular components to provide energy and building blocks, which the cell can utilize in this dire state. [12] Macroautophagy is activated as early as 30 minutes into starvation and remains at high activity for at least 4–8 hours into starvation. If the starvation state persists for more than 10 hours, the cells switch to the selective form of autophagy, namely CMA, which is known to reach a plateau of maximal activation ~36 hours into fasting and remains at these levels until ~3 days. The selectivity of CMA for individual cytosolic proteins permits cells to degrade only those proteins that might not be required in these starvation conditions in order to generate amino acids for the synthesis of essential proteins. For example, some of the best-characterized CMA substrates are enzymes involved in glycolysis, a pathway known to be less active in fasting conditions. [13] [14]
CMA is important in regulating cellular metabolism. Specific depletion of CMA in liver results in robust hepatic glycogen use accompanied with accumulation of fat in the liver, along with altered glucose homeostasis, increased energy expenditure and reduced peripheral adiposity. [14] Proteomics analyses identified several enzymes of the carbohydrate and the lipid metabolism pathways to be CMA substrates, and their altered degradation in the knockout mice explaining the abnormal metabolic phenotype of the CMA-deficient mice. [14] The ability of CMA to selectively degrade enzymes involved in the metabolism of free fatty acids (i.e. linoleic and linolic pathway) has proven key for activation of hematopoietic stem cells, [15] thus supporting a role for CMA in stem cell function. CMA activity is upregulated during differentiation of embryonic stem cells and contributed to the degradation of IDH1 and Plin2. [16] [17]
CMA activity has been shown to be modulated through retinoic acid receptor alpha signaling and is specifically activated by designed all-trans retinoic acid derivatives in cultured cells. [18]
CMA is also responsible for the selective removal of damaged and no-longer-functional proteins. This function is critical when cells are exposed to agents that cause protein damage as the selectivity of CMA ensures that only the damaged proteins get targeted to lysosomes for degradation. For instance, oxidative stress and exposure to toxic compounds are stimuli that upregulate CMA. [19] Consequently, cells that are defective for CMA are more susceptible to these insults than control cells. [20]
CMA performs various specialized functions as well, depending on the specific protein undergoing degradation through this pathway and the cell type involved. For example, known CMA substrates include, MEF2D, a neuronal factor important for survival; Pax2, a transcription factor, important for the regulation growth of renal tubular cells; IκBα, known inhibitor of NFκB. CMA has also been suggested to contribute to antigen presentation in dendritic cells. [21] [22] [23]
CMA is activated during T cell activation due to increased expression of the CMA receptor LAMP-2A. [24] CMA is essential for T cell activation through the degradation of negative regulators of T cell activation (Itch, RCAN1). Consequently, specific depletion of CMA in T cells results in immune response deficiency following immunization or infection. [24]
CMA is increased upon genotoxic stress. [25] Conversely, decreased CMA activity associates with increased genome instability and decreased cell survival. CMA is involved in the removal of Chk1, a key protein for cell cycle progression and cells with impaired CMA have defective DNA repair. [25]
CMA degrades lipid droplet proteins (perilipin 2 and perilipin 3). [26] Removal of these lipid droplet coat proteins by CMA precedes lipolysis and lipophagy. [26] Consequently, defective CMA activity leads to massive accumulation of lipid droplets and steatosis. [14] [26]
CMA activity declines with age in many cell types of old rodents and in cells of older humans. [27] [28] [29] This impairment of CMA in aging is mainly due to a decrease in the levels of LAMP-2A at the lysosomal membrane, because of reduced stability of the CMA receptor and not due to decreased de novo synthesis. Studies in a transgenic mouse model in which normal levels of LAMP-2A are maintained throughout life, showed that these animals had ‘cleaner’ cells, better response to stress – and overall, a better health-span. [29] These studies support the possible contribution of declined CMA activity to poor cellular homeostasis and inefficient response to stress characteristic of old organisms. High-fat diet inhibits CMA. [30] This is because of a decrease in the stability of the CMA receptor at the lysosomal surface. More recently CMA has been implicated in the regeneration capacity of new blood cells by sustaining hematopoietic stem cell function. [31] [32]
A primary defect in CMA activity has also been described in neurodegenerative diseases, such as Parkinson’s disease [33] [34] [35] and certain tauopathies. [36] [37] In these cases, the defect lies in the ‘tight’ binding to the lysosomal membrane of pathogenic proteins known to accumulate in these disorders (α-synuclein, UCHL1 in Parkinson’s disease and mutant Tau in tauopathies). These toxic proteins often bind to LAMP-2A with abnormal affinity exerting a ‘clogging effect’ at the lysosomal membrane and thus, inhibit the CMA-mediated degradation of other cytosolic substrate proteins. [33] [34]
Links between CMA and cancer have also been established. [38] [39] [40] CMA is upregulated in many different types of human cancer cells and blockage of CMA in these cells reduces their proliferative, tumorigenic and metastatic capabilities. In fact, interference of the expression of LAMP-2A in already-formed experimental tumors in mice resulted in their regression. [38]
A lysosome is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane proteins, and its lumenal proteins. The lumen's pH (~4.5–5.0) is optimal for the enzymes involved in hydrolysis, analogous to the activity of the stomach. Besides degradation of polymers, the lysosome is involved in various cell processes, including secretion, plasma membrane repair, apoptosis, cell signaling, and energy metabolism.
Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation or can be recycled back to the cell membrane in the endocytic cycle. Molecules are also transported to endosomes from the trans Golgi network and either continue to lysosomes or recycle back to the Golgi apparatus.
Autophagy is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. It allows the orderly degradation and recycling of cellular components. Although initially characterized as a primordial degradation pathway induced to protect against starvation, it has become increasingly clear that autophagy also plays a major role in the homeostasis of non-starved cells. Defects in autophagy have been linked to various human diseases, including neurodegeneration and cancer, and interest in modulating autophagy as a potential treatment for these diseases has grown rapidly.
In cell biology, a phagosome is a vesicle formed around a particle engulfed by a phagocyte via phagocytosis. Professional phagocytes include macrophages, neutrophils, and dendritic cells (DCs).
Heat shock 70 kDa protein 8 also known as heat shock cognate 71 kDa protein or Hsc70 or Hsp73 is a heat shock protein that in humans is encoded by the HSPA8 gene on chromosome 11. As a member of the heat shock protein 70 family and a chaperone protein, it facilitates the proper folding of newly translated and misfolded proteins, as well as stabilize or degrade mutant proteins. Its functions contribute to biological processes including signal transduction, apoptosis, autophagy, protein homeostasis, and cell growth and differentiation. It has been associated with an extensive number of cancers, neurodegenerative diseases, cell senescence, and aging.
The bafilomycins are a family of macrolide antibiotics produced from a variety of Streptomycetes. Their chemical structure is defined by a 16-membered lactone ring scaffold. Bafilomycins exhibit a wide range of biological activity, including anti-tumor, anti-parasitic, immunosuppressant and anti-fungal activity. The most used bafilomycin is bafilomycin A1, a potent inhibitor of cellular autophagy. Bafilomycins have also been found to act as ionophores, transporting potassium K+ across biological membranes and leading to mitochondrial damage and cell death.
Two-pore channels (TPCs) are eukaryotic intracellular voltage-gated and ligand gated cation selective ion channels. There are two known paralogs in the human genome, TPC1s and TPC2s. In humans, TPC1s are sodium selective and TPC2s conduct sodium ions, calcium ions and possibly hydrogen ions. Plant TPC1s are non-selective channels. Expression of TPCs are found in both plant vacuoles and animal acidic organelles. These organelles consist of endosomes and lysosomes. TPCs are formed from two transmembrane non-equivalent tandem Shaker-like, pore-forming subunits, dimerized to form quasi-tetramers. Quasi-tetramers appear very similar to tetramers, but are not quite the same. Some key roles of TPCs include calcium dependent responses in muscle contraction(s), hormone secretion, fertilization, and differentiation. Disorders linked to TPCs include membrane trafficking, Parkinson's disease, Ebola, and fatty liver.
Lysosomal lipase is a form of lipase which functions intracellularly, in the lysosomes.
Vojo Deretic, is distinguished professor and chair of the Department of Molecular Genetics and Microbiology at the University of New Mexico School of Medicine. Deretic was the founding director of the Autophagy, Inflammation and Metabolism (AIM) Center of Biomedical Research Excellence. The AIM center promotes autophagy research nationally and internationally.
Lysosome-associated membrane protein 2 (LAMP2), also known as CD107b and Mac-3, is a human gene. Its protein, LAMP2, is one of the lysosome-associated membrane glycoproteins.
Lysosomal-associated membrane protein 1 (LAMP-1) also known as lysosome-associated membrane glycoprotein 1 and CD107a, is a protein that in humans is encoded by the LAMP1 gene. The human LAMP1 gene is located on the long arm (q) of chromosome 13 at region 3, band 4 (13q34).
BAG family molecular chaperone regulator 3 is a protein that in humans is encoded by the BAG3 gene. BAG3 is involved in chaperone-assisted selective autophagy.
Lysosomal-associated transmembrane protein 4B is a protein that in humans is encoded by the LAPTM4B gene.
Mitophagy is the selective degradation of mitochondria by autophagy. It often occurs to defective mitochondria following damage or stress. The process of mitophagy was first described over a hundred years ago by Margaret Reed Lewis and Warren Harmon Lewis. Ashford and Porter used electron microscopy to observe mitochondrial fragments in liver lysosomes by 1962, and a 1977 report suggested that "mitochondria develop functional alterations which would activate autophagy." The term "mitophagy" was in use by 1998.
Proteostasis is the dynamic regulation of a balanced, functional proteome. The proteostasis network includes competing and integrated biological pathways within cells that control the biogenesis, folding, trafficking, and degradation of proteins present within and outside the cell. Loss of proteostasis is central to understanding the cause of diseases associated with excessive protein misfolding and degradation leading to loss-of-function phenotypes, as well as aggregation-associated degenerative disorders. Therapeutic restoration of proteostasis may treat or resolve these pathologies. Cellular proteostasis is key to ensuring successful development, healthy aging, resistance to environmental stresses, and to minimize homeostatic perturbations from pathogens such as viruses. Cellular mechanisms for maintaining proteostasis include regulated protein translation, chaperone assisted protein folding, and protein degradation pathways. Adjusting each of these mechanisms based on the need for specific proteins is essential to maintain all cellular functions relying on a correctly folded proteome.
Chaperone-assisted selective autophagy is a cellular process for the selective, ubiquitin-dependent degradation of chaperone-bound proteins in lysosomes.
Microautophagy is one of the three common forms of autophagic pathway, but unlike macroautophagy and chaperone-mediated autophagy, it is mediated—in mammals by lysosomal action or in plants and fungi by vacuolar action—by direct engulfment of the cytoplasmic cargo. Cytoplasmic material is trapped in the lysosome/vacuole by a random process of membrane invagination.
Ana Maria Cuervo is a Spanish-American physician, researcher, and cell biologist. She is a professor in developmental and molecular miology, anatomy and structural biology, and medicine and co-director of the Institute for Aging Studies at the Albert Einstein College of Medicine. She is best known for her research work on autophagy, the process by which cells recycle waste products, and its changes in aging and age-related diseases.
QX39 is a synthetic compound that activates chaperone-mediated autophagy (CMA) by increasing the expression of the lysosomal receptor for this pathway, LAMP2A lysosomes. It showed potent activity in vitro but has poor pharmacokinetic properties and was not suitable for animal research. Subsequent research led to the development of CA77.1, a CMA activator suitable for in vivo use.
CA77.1 (CA) is a synthetic compound that activates chaperone-mediated autophagy (CMA) by increasing the expression of the lysosomal receptor for this pathway, LAMP2A, in lysosomes. CA77.1 is a derivative of earlier compound AR7(HY-101106), which shows potent CMA activation in vitro but is not suitable for in vivo use. CA77.1 is able to activate CMA in vivo, and demonstrates brain penetrance and favorable pharmacokinetics. It has been shown in animal studies that in vivo administration of CA77.1 to enhance chaperone-mediated autophagy, may help to degrade toxic pathogenic protein products such as tau proteins and has potential applications in the treatment of Alzheimer's disease particularly in improving both behavior and neuropathology in PS19 mice models.