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Journal ArticleDOI

Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm.

22 Oct 1999-Journal of Molecular Biology (Academic Press)-Vol. 293, Iss: 2, pp 321-331
TL;DR: Many proteins that lack intrinsic globular structure under physiological conditions have now been recognized, and it appears likely that their rapid turnover, aided by their unstructured nature in the unbound state, provides a level of control that allows rapid and accurate responses of the cell to changing environmental conditions.
About: This article is published in Journal of Molecular Biology.The article was published on 1999-10-22. It has received 2804 citations till now. The article focuses on the topics: Protein structure function & Intrinsically disordered proteins.
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Journal ArticleDOI
Daniel J. Klionsky1, Kotb Abdelmohsen2, Akihisa Abe3, Joynal Abedin4  +2519 moreInstitutions (695)
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

Journal ArticleDOI
10 Jan 2002-Nature
TL;DR: The analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions, which contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
Abstract: Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

4,895 citations

Journal ArticleDOI
TL;DR: Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold, whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.
Abstract: Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold. Disordered regions can be highly conserved between species in both composition and sequence and, contrary to the traditional view that protein function equates with a stable three-dimensional structure, disordered regions are often functional, in ways that we are only beginning to discover. Many disordered segments fold on binding to their biological targets (coupled folding and binding), whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.

3,599 citations

Journal ArticleDOI
15 Nov 2000-Proteins
TL;DR: Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins and shows that “natively unfolded” proteins are specifically localized within a unique region of charge‐hydrophobia phase space.
Abstract: "Natively unfolded" proteins occupy a unique niche within the protein kingdom in that they lack ordered structure under conditions of neutral pH in vitro. Analysis of amino acid sequences, based on the normalized net charge and mean hydrophobicity, has been applied to two sets of proteins: small globular folded proteins and "natively unfolded" ones. The results show that "natively unfolded" proteins are specifically localized within a unique region of charge-hydrophobicity phase space and indicate that a combination of low overall hydrophobicity and large net charge represent a unique structural feature of "natively unfolded" proteins.

2,029 citations


Cites background from "Intrinsically unstructured proteins..."

  • ...Moreover, a disorder-order transition induced in “natively unfolded” proteins during the binding of specific targets in vivo might represent a simple mechanism for regulation of numerous cellular processes, including transcriptional and translational regulation and cell cycle control.(29) Evolutionary persistence of the “natively unfolded” proteins represents additional confirmation of their importance and raises intriguing questions on the role of protein disorders in biologic processes....

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  • ...It has been suggested that the lack of rigid globular structure under physiologic conditions might represent a considerable functional advantage for “natively unfolded” proteins, as their large plasticity allows them to interact efficiently with several different targets.(29) Moreover, a disorder-order transition induced in “natively unfolded” proteins during the binding of specific targets in vivo might represent a simple mechanism for regulation of numerous cellular processes, including transcriptional and translational regulation and cell cycle control....

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Journal ArticleDOI
TL;DR: In this review, recent findings are surveyed to illustrate that this novel but rapidly advancing field has reached a point where proteins can be comprehensively classified on the basis of structure and function.

2,027 citations

References
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Journal ArticleDOI
29 Nov 1996-Cell
TL;DR: It is demonstrated that p300/CBP acetylates nucleosomes in concert with PCAF, a novel class of acetyltransferases in that it does not have the conserved motif found among various other acetyl transferases.

2,863 citations


"Intrinsically unstructured proteins..." refers background in this paper

  • ...In addition, there are HAT and bromodomains associated with chromatin remodeling (Ogryzko et al., 1996; Jeanmougin et al., 1997) and putative PHD and ZZ zinc ®nger domains of unknown function (Aasland et al....

    [...]

  • ...In addition, there are HAT and bromodomains associated with chromatin remodeling (Ogryzko et al., 1996; Jeanmougin et al., 1997) and putative PHD and ZZ zinc ®nger domains of unknown function (Aasland et al., 1995; Ponting et al., 1996)....

    [...]

Journal ArticleDOI
23 Dec 1998-Cell
TL;DR: Crystal structures of the human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) and the OHT-LBD complex reveal the two distinct mechanisms by which structural features of OHT promote this "autoinhibitory" helix 12 conformation.

2,581 citations


"Intrinsically unstructured proteins..." refers background in this paper

  • ...…domain includes the LXXLL motif that has been directly implicated in nuclear receptor binding (Torchia et al., 1997; Heery et al., 1997), and which has been shown, for other coactivators, to form an a-helix upon complex formation (Nolte et al., 1998; Darimont et al., 1998; Shiau et al., 1998)....

    [...]

  • ..., 1997), and which has been shown, for other coactivators, to form an a-helix upon complex formation (Nolte et al., 1998; Darimont et al., 1998; Shiau et al., 1998)....

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Journal ArticleDOI
24 Sep 1998-Nature
TL;DR: The X-ray crystal structure of a core synaptic fusion complex containing syntaxin-1A, synaptobrevin-II and SNAP-25B reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins.
Abstract: The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

2,381 citations


"Intrinsically unstructured proteins..." refers background in this paper

  • ...An X-ray structure has been determined for the core synaptic SNARE complex formed by syntaxin-1A, synaptobrevin-II, and SNAP-25B; the ternary complex consists of a twisted four-helix bundle 120 AÊ in length (Sutton et al., 1998)....

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Journal ArticleDOI
TL;DR: It is argued that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, and it is likely that this conformational transition is a fundamental event in the propagation of prions.
Abstract: Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.

2,230 citations

Journal ArticleDOI
12 Jun 1997-Nature
TL;DR: It is proposed that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.
Abstract: The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes1,2. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160 (3-5), TIF-2/GRIP-1 (refs 6, 7) and CBP/p300 (refs 4, 5, 8, 9) which function as co-activators of transcription, and RIP-140 (ref. 10), TIF-1 (ref. 11) and TRIP-1/SUG-1 (refs 12, 13) whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function14. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.

2,166 citations


"Intrinsically unstructured proteins..." refers background in this paper

  • ...…receptor interaction domain includes the LXXLL motif that has been directly implicated in nuclear receptor binding (Torchia et al., 1997; Heery et al., 1997), and which has been shown, for other coactivators, to form an a-helix upon complex formation (Nolte et al., 1998; Darimont et…...

    [...]

  • ...The unstructured receptor interaction domain includes the LXXLL motif that has been directly implicated in nuclear receptor binding (Torchia et al., 1997; Heery et al., 1997), and which has been shown, for other coactivators, to form an a-helix upon complex formation (Nolte et al....

    [...]