Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

Ryhänen, Tuomas; Hyttinen, Juha M. T.; Kopitz, Juergen; Rilla, Kirsi; Kuusisto, Erkki; Mannermaa, Eliisa; Viiri, Johanna; Holmberg, Carina I.; Immonen, Ilkka; Meri, Seppo; Parkkinen, Jussi; Eskelinen, Eeva‐Liisa; Uusitalo, Hannu; Salminen, Antero; Kaarniranta, Kai

doi:10.1111/j.1582-4934.2008.00577.x

Cited by 122 publications

(150 citation statements)

References 50 publications

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“…Cells were exposed to 5 mM MG132 for 24 h to induce the formation of perinuclear aggregates. 185 The cells were then exposed to a UV pulse (the UV-induced area is shown by red lines that are inside of the nucleus) that converts Dendra2 from green to red, and the time shown after the pulse is indicated. SQSTM1/p62 is present in a small nuclear aggregrate, and is shuttled from the nucleus to a perinuclear large protein aggregate (detected as red).…”

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confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

Self Cite

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“…The two conjugation systems are proposed to function during elongation and expansion of the phagophore membrane (14,19,22,23). A conservative estimate of the autophagy network counts over 400 proteins, which, besides the ATG-proteins, also include stress-response factors, cargo adaptors, and chaperones such as p62/SQSTM1 and heat shock protein 70 (HSP70) (15,19,22,24,(26)(27)(28).…”

Section: Introductionmentioning

confidence: 99%

Autophagy-Mediated Defense Response of Mouse Mesenchymal Stromal Cells (MSCs) to Challenge with Escherichia coli

Gorbunov¹,

Garrison²,

Zhai³

et al. 2012

Protein Interactions

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“…UPP is the master proteolytic system in the cells that clean misfolded Ub-labelled soluble proteins. Moreover, lysosomal autophagy is integrated with proteasomal degradation system (Ryhänen et al, 2009;Viiri et al, 2013). In this study, we show that Hsp70 is accumulated in both basal epithelium and stromal cells.…”

Section: Discussionmentioning

confidence: 52%

“…The ubiquitin-associated domain (UBA) of SQSTM1/p62 enables a non-covalent binding to ubiquitin or ubiquitinated substrate proteins that are then targeted to proteasomal degradation, where the N-terminal PB1 domain of p62 binds to the proteasome (Seibenhener et al, 2004;Babu et al, 2005). Alternatively, ubiquitinated complexes are shuttled to autophagocytic degradation (Ryhänen et al, 2009;Viiri et al, 2013;Komatsu et al, 2007;Pankiv et al, 2007;Kaarniranta et al, 2013). Decreased proteasomal activity evokes Hsp70, Ub protein conjugates and SQSTM1/p62 accumulation in cells (Ryhänen et al, 2009;Viiri et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

A novel proteotoxic stress‐associated mechanism for macular corneal dystrophy

Szalai

Smędowski²,

Wylęgała³

et al. 2014

Acta Ophthalmologica

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Summary. Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corneal stroma. Abnormal accumulation of proteoglycan aggregates has been observed intra-and extracellularly in the stromal layer. In addition to the stromal keratocytes and corneal lamellae, deposits are also present in the basal epithelial cells, endothelial cells and Descemet's membrane. Misfolding of proteins has a tendency to gather into aggregating deposits. We studied interaction of molecular chaperones and proteasomal clearance in macular dystrophy human samples and in human corneal HCE-2 epithelial cells. Seven cases of macular corneal dystrophy and four normal corneal buttons collected during corneal transplantation were examined for their expression patterns of heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62. In response to proteasome inhibition the same proteins were analyzed by western blotting. Slitlamp examination, in vivo confocal cornea microscopy and transmission electron microscopy were used for morphological analyses. Heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62 were upregulated in both the basal corneal epithelial cells and the stromal keratocytes in macular corneal dystrophy samples that coincided with an increased expression of the same molecules under proteasome inhibition in the HCE-2 cells in vitro. We propose a novel regulatory mechanism that connects the molecular chaperone and proteasomal clearance system in the pathogenesis of macular corneal dystrophy.

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Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

Cited by 122 publications

References 50 publications

Guidelines for the use and interpretation of assays for monitoring autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy

Autophagy-Mediated Defense Response of Mouse Mesenchymal Stromal Cells (MSCs) to Challenge with Escherichia coli

A novel proteotoxic stress‐associated mechanism for macular corneal dystrophy

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