Chaperone-mediated autophagy in protein quality control

Chaperone-mediated autophagy is a selective mechanism for degradation of soluble cytosolic proteins in lysosomes that distinguishes itself from other autophagic pathways by the selectivity with which CMA substrates are targeted for degradation. The recent molecular dissection of this autophagic pathway and the development of experimental models with compromised CMA have unveiled the important contribution of this pathway to protein quality control. In fact, CMA activation seems to be a common mechanism of cellular defense against proteotoxicity.     

Chaperone-mediated autophagy in health and disease

Chaperone-mediated autophagy (CMA) is a lysosomal pathway that participates in the degradation of cytosolic proteins. CMA is activated by starvation and in response to stressors that result in protein damage. The selectivity intrinsic to CMA allows for removal of damaged proteins without disturbing nearby functional ones. CMA works in a coordinated manner with other autophagic pathways, which can compensate for each other. Interest in CMA has recently grown because of the connections established between this autophagic pathway and human pathologies. Here we review the unique properties of CMA compared to other autophagic pathways and its relevance in health and disease.     

Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance

Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway for the degradation of cytosolic proteins. We review in this work some of the recent findings on this pathway regarding the molecular mechanisms that contribute to substrate targeting, binding and translocation across the lysosomal membrane. We have placed particular emphasis on the critical role that changes in the lipid composition of the lysosomal membrane play in the regulation of CMA, as well as the modulatory effect of other novel CMA components. In the second part of this review, we describe the physiological relevance of CMA and its role as one of the cellular mechanisms involved in the response to stress. Changes with age in CMA activity and the contribution of failure of CMA to the phenotype of aging and to the pathogenesis of several age-related pathologies are also described.     

Chaperone-mediated autophagy: Dice's 'wild' idea about lysosomal selectivity

A little over 1 year ago, we lost a bright scientist and a dear colleague who, in his younger years, proposed the 'heretical' idea that lysosomes could selectively degrade cytosolic proteins. That scientist was J. Fred Dice, and his lifetime's discovery was the degradative pathway that we now know as chaperone-mediated autophagy.     

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome

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Chaperone-mediated autophagy prevents apoptosis by degrading BBC3/PUMA

Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that dictate the cell fate choice between autophagy and apoptosis remain largely unknown. Here we report that the proapoptotic protein BBC3/PUMA (BCL2 binding component 3) is a bona fide substrate of chaperone-mediated autophagy (CMA). BBC3 associates with HSPA8/HSC70 (heat shock 70kDa protein 8), leading to its lysosome translocation and uptake. Inhibition of CMA results in stabilization of BBC3, which in turn sensitizes tumor cells to undergo apoptosis. We further demonstrate that upon TNF (tumor necrosis factor) treatment, IKBKB/IKKβ (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β)-mediated BBC3 Ser10 phosphorylation is crucial for BBC3 stabilization via blocking its degradation by CMA. Mechanistically, Ser10 phosphorylation facilitates BBC3 translocation from the cytosol to mitochondria. BBC3 stabilization resulting from either Ser10 phosphorylation or CMA inhibition potentiates TNF-induced apoptotic cell death. Our findings thus reveal that the selective degradation of BBC3 underlies the prosurvival role of CMA and define a previously unappreciated proapoptotic role of IKBKB that acts through phosphorylation-mediated stabilization of BBC3, thereby promoting TNF-triggered apoptosis.     

Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

Chaperone-mediated autophagy (CMA), a selective form of protein lysosomal degradation, is maximally activated in stress situations to ensure maintenance of cellular homeostasis. CMA activity decreases with age and in several human chronic disorders, but in contrast, in most cancer cells, CMA is upregulated and required for tumor growth. However, the role of CMA in malignant transformation remains unknown. In this study, we demonstrate that CMA inhibition in fibroblasts augments the efficiency of MYC/c-Myc-driven cellular transformation. CMA blockage contributes to the increase of total and nuclear MYC, leading to enhancement of cell proliferation and colony formation. Impaired CMA functionality accentuates tumorigenesis-related metabolic changes observed upon MYC-transformation. Although not a direct CMA substrate, we have found that CMA regulates cellular MYC levels by controlling its proteasomal degradation. CMA promotes MYC ubiquitination and degradation by regulating the degradation of C330027C09Rik/KIAA1524/CIP2A (referred to hereafter as CIP2A), responsible for MYC stabilization. Ubiquitination and proteasomal degradation of MYC requires dephosphorylation at Ser62, and CIP2A inhibits the phosphatase responsible for this dephosphorylation. Failure to degrade CIP2A upon CMA blockage leads to increased levels of phosphorylated MYC (Ser62) and to stabilization of this oncogene. We demonstrate that this phosphorylation is essential for the CMA-mediated effect, since specific mutation of this site (Ser62 to Ala62) is enough to normalize MYC levels in CMA-incompetent cells. Altogether these data demonstrate that CMA mitigates MYC oncogenic activity by promoting its proteasomal degradation and reveal a novel tumor suppressive role for CMA in nontumorigenic cells.     

Chaperone-mediated autophagy and aging: a novel regulatory role of lipids revealed

A wide pool of cytosolic proteins is selectively degraded in lysosomes by chaperonemediated autophagy (CMA). Binding of these proteins to a receptor at the lysosomal membrane is the limiting step in CMA. Levels of this receptor are tightly regulated through changes in its degradation, multimeric organization and dynamic distribution between the lysosomal membrane and lumen. We have now reported that subcompartmentalization of the receptor in discrete lipid microdomains at the lysosomal membrane regulates its engagement in each of these processes-degradation, multimerization and membrane retrieval. Changes in the lipid composition of the membrane thus affect the dynamics of the receptor and, consequently, CMA activity. As an example of CMA dysfunction resulting from perturbation of the lipid composition of the lysosomal membrane, we discuss here a second study in which we analyzed the changes in the dynamics of the receptor during aging. CMA activity decreases with age primarily due to a decrease in the levels of the CMA receptor at the lysosomal membrane. Now we have found that age-related alterations in the lipid composition of the discrete microdomains at the lysosomal membrane are behind the reduced lysosomal levels of the receptor and, consequently, the declined CMA activity that occurs during aging.     

Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer

LAMP2A is the key protein of chaperone-mediated autophagy (CMA), downregulation of LAMP2A leads to CMA blockade. CMA activation has been implicated in cancer growth, but the exact mechanisms are unclear. Elevated expression of LAMP2A was found in 8 kinds of tumors (n=747), suggesting that LAMP2A may have an important role in cancer progression. Unsurprisingly, LAMP2A knockdown in gastric cancer (GC) cells hindered proliferation, accompanied with altered expression of cell cycle-related proteins and accumulation of RND3/RhoE. Interactomic and KEGG analysis revealed that RND3 was a putative CMA substrate. Further study demonstrated that RND3 silencing could partly rescue the proliferation arrest induced by LAMP2A knockdown; RND3 was increased upon lysosome inhibition via both chemicals and LAMP2A-shRNA; Furthermore, RND3 could interact with CMA components HSPA8 and LAMP2A, and be engulfed by isolated lysosomes. Thus, constant degradation of RND3 by CMA is required to sustain rapid proliferation of GC cells. At last, the clinical significance of LAMP2A was explored in 593 gastric noncancerous lesions and 173 GC tissues, the results revealed that LAMP2A is a promising biomarker for GC early warning and prognosis of female GC patients.     

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal

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Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Chaperone-mediated inhibition of tubulin self-assembly

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.     

Chaperone-mediated in vitro disassembly of polyoma- and papillomaviruses

Hsp70 chaperones play a role in polyoma- and papillomavirus assembly, as evidenced by their interaction in vivo with polyomavirus capsid proteins at late times after virus infection and by their ability to assemble viral capsomeres into capsids in vitro. We studied whether Hsp70 chaperones might also participate in the uncoating reaction. In vivo, Hsp70 co-immunoprecipitated with polyomavirus virion VP1 at 3 h after infection of mouse cells. In vitro, prokaryotic and eukaryotic Hsp70 chaperones efficiently disassembled polyoma- and papillomavirus-like particles and virions in energy-dependent reactions. These observations support a role for cell chaperones in the disassembly of these viruses.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.     

Chaperone-mediated regulation of hepatic protein secretion by caloric restriction

Calorie restriction (CR) delays age-related physiological changes, reduces cancer incidence, and increases maximum life span in mammals. Here we show that CR decreased the expression of many hepatic molecular chaperones and concomitantly increased the rate and efficiency of serum protein secretion. Hepatocytes from calorie-restricted mice secreted twice as much albumin, 63% more alpha1-antitrypsin, and 250% more of the 31.5-kDa protein 2 h after their synthesis. A number of trivial explanations for these results, such as differential rates of protein synthesis and cell leakage during the assay, were eliminated. These novel results suggest that CR may promote the secretion of serum proteins, thereby promoting serum protein turnover. This may reduce the circulating level of damaging, glycoxidated serum proteins.