Assessment of mammalian endosomal microautophagy

Krause, Gregory J.; Cuervo, Ana María

doi:10.1016/bs.mcb.2020.10.009

Cited by 12 publications

(19 citation statements)

References 37 publications

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“…Previous data showed that LE/MVBs from old animals accumulate undegraded oxidized proteins (Cannizzo et al, 2012 ), suggesting a possible age‐related decline in eMI function. To investigate this possibility, we isolated LE/MVBs from young (4–6 m) and old (22–24 m) mouse livers and reconstituted the different steps of eMI in vitro using a previously developed assay (Krause & Cuervo, 2021 ; Sahu et al, 2011 ) (Figure 1a ; comparable purity of fractions isolated from young and old mice was verified by immunoblot for LE/MVBs markers and enzymatic assays for the endolysosomal enzyme β‐hexosaminidase [Figure S1 a–c]). LE/MVBs were incubated with two KFERQ‐motif containing proteins (Tau and α‐synuclein), previously shown to undergo degradation by selective eMI (Caballero et al, 2018 ; Krause et al, 2022 ) or with Cyclophilin A, which lacks a KFERQ motif and can be internalized and degraded in LE/MVBs through non‐selective eMI (Sahu et al, 2011 ) (Figure 1b ).…”

Section: Resultsmentioning

confidence: 99%

“…To identify possible molecular mediators of the proposed coordinated functioning of eMI and exocytosis, we performed a CRISPR interference (CRISPRi) screen in cells expressing a nuclease‐dead version of the Cas9 enzyme (dCas9) tagged with the KRAB transcriptional repressor and transduced for stable expression of the KFERQ‐split Venus eMI reporter (Figure S5a ). This reporter allows visualization of the sequestration of this artificial eMI substrate in the inward budding vesicles at the LE/MVB limiting membrane (Caballero et al, 2018 ; Krause & Cuervo, 2021 ). The reporter only fluoresces when the two halves of the Venus protein come in close proximity in the confined space of the intraluminal vesicles of LE/MVBs, making measurement of the number of fluorescent puncta per cell a reliable measurement of eMI.…”

Section: Resultsmentioning

confidence: 99%

“…We instead considered changes in RalA degradation with age and noticed the presence of a KFERQ-like motif in its sequence ( 197 KRIRE 201 becomes a motif upon acetylation of the lysine residue). Since our proteomic analysis (Figure 2 and S7f) did not support degradation of RalA in LE/MVBs and given that KFERQ pentapeptides are shared as targeting motifs for both eMI and CMA (Krause & Cuervo, 2021), we investigated if RalA could be degraded by CMA.…”

Section: Age-related Changes In Exocyst Complex Organization In Le/mvbsmentioning

confidence: 99%

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Reduced endosomal microautophagy activity in aging associates with enhanced exocyst‐mediated protein secretion

et al. 2022

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Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age‐related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub‐proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age‐related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein‐loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst‐RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst‐RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst‐RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Age-related Changes In Exocyst Complex Organization In Le/mvbsmentioning

confidence: 99%

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Reduced endosomal microautophagy activity in aging associates with enhanced exocyst‐mediated protein secretion

et al. 2022

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“…CMA can be a potential target to regulate the function of hematopoietic stem cells and is integral to the proper functioning of part of the normal function of hematopoietic stem cells ( Dong et al, 2021 ). Endosomal microautophagy, a selective autophagy that occurs through endosomal autophagy, and HSC70 chaperones specifically bind to the KFERQ-like motif partially similar to CMA ( Tekirdag and Cuervo, 2018 ; Krause and Cuervo, 2021 ).…”

Section: Classification Of Autophagymentioning

confidence: 99%

Research progress on the relationship between autophagy and chronic complications of diabetes

Wang

Fei

et al. 2022

Front. Physiol.

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Diabetes is a common metabolic disease whose hyperglycemic state can induce diverse complications and even threaten human health and life security. Currently, the treatment of diabetes is restricted to drugs that regulate blood glucose and have certain accompanying side effects. Autophagy, a research hotspot, has been proven to be involved in the occurrence and progression of the chronic complications of diabetes. Autophagy, as an essential organismal defense mechanism, refers to the wrapping of cytoplasmic proteins, broken organelles or pathogens by vesicles, which are then degraded by lysosomes to maintain the stability of the intracellular environment. Here, we review the relevant aspects of autophagy and the molecular mechanisms of autophagy in diabetic chronic complications, and further analyze the impact of improving autophagy on diabetic chronic complications, which will contribute to a new direction for further prevention and treatment of diabetic chronic complications.

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“…Effective macroautophagy is often referred to as "cell cleansing", and, when of moderate intensity, and appropriately balanced by synthesis of new proteins and organelles-particularly mitochondria, produced in the complex process of "mitochondrial biogenesis"-it is generally thought to enhance health span [1][2][3][4]. (Henceforth, macroautophagy will be referred to simply as "autophagy"-albeit chaperone-mediated autophagy and microautophagy are distinct processes that also assist in protein disposal within autophagosomes [5,6].) Measures which are known to increase median and maximal lifespan in rodents typically are associated with increased autophagy.…”

Section: Boosting Autophagy For Health Protectionmentioning

confidence: 99%

Nutraceutical and Dietary Strategies for Up-Regulating Macroautophagy

McCarty

2022

IJMS

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Macroautophagy is a “cell cleansing” process that rids cells of protein aggregates and damaged organelles that may contribute to disease pathogenesis and the dysfunctions associated with aging. Measures which boost longevity and health span in rodents typically up-regulate macroautophagy, and it has often been suggested that safe strategies which can promote this process in humans may contribute to healthful aging. The kinase ULK1 serves as a trigger for autophagy initiation, and the transcription factors TFEB, FOXO1, ATF4 and CHOP promote expression of a number of proteins which mediate macroautophagy. Nutraceutical or dietary measures which stimulate AMPK, SIRT1, eIF5A, and that diminish the activities of AKT and mTORC1, can be expected to boost the activities of these pro-autophagic factors. The activity of AMPK can be stimulated with the phytochemical berberine. SIRT1 activation may be achieved with a range of agents, including ferulic acid, melatonin, urolithin A, N1-methylnicotinamide, nicotinamide riboside, and glucosamine; correction of ubiquinone deficiency may also be useful in this regard, as may dietary strategies such as time-restricted feeding or intermittent fasting. In the context of an age-related decrease in cellular polyamine levels, provision of exogenous spermidine can boost the hypusination reaction required for the appropriate post-translational modification of eIF5A. Low-protein plant-based diets could be expected to increase ATF4 and CHOP expression, while diminishing IGF-I-mediated activation of AKT and mTORC1. Hence, practical strategies for protecting health by up-regulating macroautophagy may be feasible.

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Assessment of mammalian endosomal microautophagy

Cited by 12 publications

References 37 publications

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst‐mediated protein secretion

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst‐mediated protein secretion

Research progress on the relationship between autophagy and chronic complications of diabetes

Nutraceutical and Dietary Strategies for Up-Regulating Macroautophagy

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