Aggresomes: A Cellular Response to Misfolded Proteins
28 Dec 1998-Journal of Cell Biology (The Rockefeller University Press)-Vol. 143, Iss: 7, pp 1883-1898
TL;DR: The intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR) is investigated and it is demonstrated that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which is termed the aggresome.
Abstract: Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.
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TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: 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.
For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.
5,187 citations
TL;DR: It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease.
Abstract: Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, conside...
3,990 citations
Cites background from "Aggresomes: A Cellular Response to ..."
...The proteasome interacts with components of the cell cycle machinery (216), and proteasomal subunits colocalize in vivo together with heat shock proteins and chaperones at sites of misfolded AR aggregates (213, 421)....
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...While the initial hypothesis was that inclusion bodies are generated because of the inherent tendency of the abnormal proteins to associate with one another and aggregate, it is now thought that the process may be more complex and involves active cellular machineries (99, 212, 213), including inhibition of the ubiquitin system by the aggregated proteins (25)....
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TL;DR: A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.
Abstract: The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.
2,004 citations
Cites background from "Aggresomes: A Cellular Response to ..."
...As abnormal proteins accumulate, larger inclusions are found near the centrosome as a result of an active microtubule-dependent process that isolates the unfolded proteins in amorphous structures, which are often termed 'aggresomes...
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01 Jan 2003TL;DR: It is reported that protein aggregation directly impaired the function of the ubiquitin-proteasome system, suggesting a potential mechanism linking protein aggregation to cellular disregulation and cell death.
Abstract: Intracellular deposition of aggregated and ubiquitylated proteins is a prominent cytopathological feature of most neurodegenerative disorders. Whether protein aggregates themselves are pathogenic or are the consequence of an underlying molecular lesion is unclear. Here, we report that protein aggregation directly impaired the function of the ubiquitin-proteasome system. Transient expression of two unrelated aggregation-prone proteins, a huntingtin fragment containing a pathogenic polyglutamine repeat and a folding mutant of cystic fibrosis transmembrane conductance regulator, caused nearly complete inhibition of the ubiquitin-proteasome system. Because of the central role of ubiquitin-dependent proteolysis in regulating fundamental cellular events such as cell division and apoptosis, our data suggest a potential mechanism linking protein aggregation to cellular disregulation and cell death.
1,997 citations
TL;DR: This work has suggested that, in animal cells, aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent retrograde transport on microtubules and this microtubule-dependent inclusion body is called an aggresome.
1,895 citations
References
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TL;DR: A new technique for assaying infectivity of adenovirus 5 DNA has been developed and a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated.
9,230 citations
"Aggresomes: A Cellular Response to ..." refers methods in this paper
...Transient transfections were carried out by adding plasmid DNA as a calcium phosphate precipitate (Graham and Eb, 1973)....
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TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients
6,731 citations
"Aggresomes: A Cellular Response to ..." refers background in this paper
...CFTR is an z 165-kD polytopic glycoprotein which encodes a Cl 2 ion channel in the plasma membrane of epithelial cells (Riordan et al., 1989)....
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TL;DR: Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.
Abstract: The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.
3,522 citations
"Aggresomes: A Cellular Response to ..." refers background in this paper
...Molecular chaperones bind nonnative protein conformations, sequestering misfolded protein or aggregation-prone folding intermediates from the cytosol, thereby reducing the likelihood of aggregate formation (Hartl, 1996)....
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...Binding of molecular chaperones like Hsp70 or Hsp90 to surface-exposed hydrophobic domains on nascent or misfolded proteins can serve to minimize protein aggregation (Hartl, 1996)....
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TL;DR: A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
2,946 citations
"Aggresomes: A Cellular Response to ..." refers background in this paper
...CFTR is an z 165-kD polytopic glycoprotein which encodes a Cl 2 ion channel in the plasma membrane of epithelial cells (Riordan et al., 1989)....
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TL;DR: Cystic fibrosis is a regulated Cl- channel, for which structure-function relationships have begun to be established, and insight into the functions of individual domains has come from a number of studies.
1,446 citations