Intrinsically disordered regions in autophagy proteins

Autophagy is an essential eukaryotic pathway required for cellular homeostasis. Numerous key autophagy effectors and regulators have been identified, but the mechanism by which they carry out their function in autophagy is not fully understood. Our rigorous bioinformatic analysis shows that the majority of key human autophagy proteins include intrinsically disordered regions (IDRs), which are sequences lacking stable secondary and tertiary structure; suggesting that IDRs play an important, yet hitherto uninvestigated, role in autophagy. Available crystal structures corroborate the absence of structure in some of these predicted IDRs. Regions of orthologs equivalent to the IDRs predicted in the human autophagy proteins are poorly conserved, indicating that these regions may have diverse functions in different homologs. We also show that IDRs predicted in human proteins contain several regions predicted to facilitate protein–protein interactions, and delineate the network of proteins that interact with each predicted IDR‐containing autophagy protein, suggesting that many of these interactions may involve IDRs. Lastly, we experimentally show that a BCL2 homology 3 domain (BH3D), within the key autophagy effector BECN1 is an IDR. This BH3D undergoes a dramatic conformational change from coil to α‐helix upon binding to BCL2s, with the C‐terminal half of this BH3D constituting a binding motif, which serves to anchor the interaction of the BH3D to BCL2s. The information presented here will help inform future in‐depth investigations of the biological role and mechanism of IDRs in autophagy proteins. Proteins 2014; 82:565–578. © 2013 Wiley Periodicals, Inc.     

Intrinsically disordered proteins as molecular shields

The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by F?rster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.     

Intrinsically disordered proteins: regulation and disease

Intrinsically disordered proteins (IDPs) are enriched in signaling and regulatory functions because disordered segments permit interaction with several proteins and hence the re-use of the same protein in multiple pathways. Understanding IDP regulation is important because altered expression of IDPs is associated with many diseases. Recent studies show that IDPs are tightly regulated and that dosage-sensitive genes encode proteins with disordered segments. The tight regulation of IDPs may contribute to signaling fidelity by ensuring that IDPs are available in appropriate amounts and not present longer than needed. The altered availability of IDPs may result in sequestration of proteins through non-functional interactions involving disordered segments (i.e., molecular titration), thereby causing an imbalance in signaling pathways. We discuss the regulation of IDPs, address implications for signaling, disease and drug development, and outline directions for future research.     

Intrinsically disordered proteins from A to Z

The ideas that proteins might possess specific functions without being uniquely folded into rigid 3D-structures and that these floppy polypeptides might constitute a noticeable part of any given proteome would have been considered as a preposterous fiction 15 or even 10 years ago. The situation has changed recently, and the existence of functional yet intrinsically disordered proteins and regions has become accepted by a significant number of protein scientists. These fuzzy objects with fuzzy structures and fuzzy functions are among the most interesting and attractive targets for modern protein research. This review summarizes some of the major discoveries and breakthroughs in the field of intrinsic disorder by representing related concepts and definitions.     

Intrinsically disordered proteins: modes of binding with emphasis on disordered domains

Our notions of protein function have long been determined by the protein structure–function paradigm. However, the idea that protein function is dictated by a prerequisite complementarity of shapes at the binding interface is becoming increasingly challenged. Interactions involving intrinsically disordered proteins (IDPs) have indicated a significant degree of disorder present in the bound state, ranging from static disorder to complete disorder, termed ‘random fuzziness’. This review assesses the anatomy of an IDP and relates how its intrinsic properties permit promiscuity and allow for the various modes of interaction. Furthermore, a mechanistic overview of the types of disordered domains is detailed, while also relating to a recent example and the kinetic and thermodynamic principles governing its formation.     

Intrinsically disordered proteins and biomolecular condensates as drug targets

Intrinsically disordered domains represent attractive therapeutic targets because they play key roles in cancer, as well as in neurodegenerative and infectious diseases. They are, however, considered undruggable because they do not form stable binding pockets for small molecules and, therefore, have not been prioritized in drug discovery. Under physiological solution conditions many biomedically relevant intrinsically disordered proteins undergo phase separation processes leading to the formation of mesoscopic highly dynamic assemblies, generally known as biomolecular condensates that define environments that can be quite different from the solutions surrounding them. In what follows, we review key recent findings in this area and show how biomolecular condensation can offer opportunities for modulating the activities of intrinsically disordered targets.     

Intrinsically disordered regions have specific functions in mitochondrial and nuclear proteins

Proteins in general consist not only of globular structural domains (SDs), but also of intrinsically disordered regions (IDRs), i.e. those that do not assume unique three-dimensional structures by themselves. Although IDRs are especially prevalent in eukaryotic proteins, the functions are mostly unknown. To elucidate the functions of IDRs, we first divided eukaryotic proteins into subcellular localizations, identified IDRs by the DICHOT system that accurately divides entire proteins into SDs and IDRs, and examined charge and hydropathy characteristics. On average, mitochondrial proteins have IDRs more positively charged than SDs. Comparison of mitochondrial proteins with orthologous prokaryotic proteins showed that mitochondrial proteins tend to have segments attached at both N and C termini, high fractions of which are IDRs. Segments added to the N-terminus of mitochondrial proteins contain not only signal sequences but also mature proteins and exhibit a positive charge gradient, with the magnitude increasing toward the N-terminus. This finding is consistent with the notion that positively charged residues are added to the N-terminus of proteobacterial proteins so that the extended proteins can be chromosomally encoded and efficiently transported to mitochondria after translation. By contrast, nuclear proteins generally have positively charged SDs and negatively charged IDRs. Among nuclear proteins, DNA-binding proteins have enhanced charge tendencies. We propose that SDs in nuclear proteins tend to be positively charged because of the need to bind to negatively charged nucleotides, while IDRs tend to be negatively charged to interact with other proteins or other regions of the same proteins to avoid premature proteasomal degradation.     

Intrinsically disordered proteins display no preference for chaperone binding in vivo

Intrinsically disordered/unstructured proteins (IDPs) are extremely sensitive to proteolysis in vitro, but show no enhanced degradation rates in vivo. Their existence and functioning may be explained if IDPs are preferentially associated with chaperones in the cell, which may offer protection against degradation by proteases. To test this inference, we took pairwise interaction data from high-throughput interaction studies and analyzed to see if predicted disorder correlates with the tendency of chaperone binding by proteins. Our major finding is that disorder predicted by the IUPred algorithm actually shows negative correlation with chaperone binding in E. coli, S. cerevisiae, and metazoa species. Since predicted disorder positively correlates with the tendency of partner binding in the interactome, the difference between the disorder of chaperone-binding and non-binding proteins is even more pronounced if normalized to their overall tendency to be involved in pairwise protein–protein interactions. We argue that chaperone binding is primarily required for folding of globular proteins, as reflected in an increased preference for chaperones of proteins in which at least one Pfam domain exists. In terms of the functional consequences of chaperone binding of mostly disordered proteins, we suggest that its primary reason is not the assistance of folding, but promotion of assembly with partners. In support of this conclusion, we show that IDPs that bind chaperones also tend to bind other proteins.     

Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

RNA-binding proteins play crucial roles in various cellular functions and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15), as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3. Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, whereas underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts.     

固有无序蛋白研究进展及其对细胞胁迫耐受性的影响

固有无序蛋白(intrinsically disordered proteins,IDPs)是指在生理条件下缺乏有序稳定的高级结构,整体或局部不折叠,但能够参与多种生物学过程、行使特定生物学功能的一类蛋白质.固有无序蛋白决定了其不同于经典蛋白质"序列-结构-功能"的功能范式,丰富了蛋白质"结构-功能"的多样性.固有无序...     

Molecular principles of the interactions of disordered proteins

Thorough knowledge of the molecular principles of protein-protein recognition is essential to our understanding of protein function at the cellular level. Whereas interactions of ordered proteins have been analyzed in great detail, complexes of intrinsically unstructured/disordered proteins (IUPs) have hardly been addressed so far. Here, we have collected a database of 39 complexes of experimentally verified IUPs, and compared their interfaces with those of 72 complexes of ordered, globular proteins. The characteristic differences found between the two types of complexes suggest that IUPs represent a distinct molecular implementation of the principles of protein-protein recognition. The interfaces do not differ in size, but those of IUPs cover a much larger part of the surface of the protein than for their ordered counterparts. Moreover, IUP interfaces are significantly more hydrophobic relative to their overall amino acid composition, but also in absolute terms. They rely more on hydrophobic-hydrophobic than on polar-polar interactions. Their amino acids in the interface realize more intermolecular contacts, which suggests a better fit with the partner due to induced folding upon binding that results in a better adaptation to the partner. The two modes of interaction also differ in that IUPs usually use only a single continuous segment for partner binding, whereas the binding sites of ordered proteins are more segmented. Probably, all these features contribute to the increased evolutionary conservation of IUP interface residues. These noted molecular differences are also manifested in the interaction energies of IUPs. Our approximation of these by low-resolution force-fields shows that IUPs gain much more stabilization energy from intermolecular contacts, than from folding, i.e. they use their binding energy for folding. Overall, our findings provide a structural rationale to the prior suggestions that many IUPs are specialized for functions realized by protein-protein interactions.     

Intrinsically disordered regions of human plasma membrane proteins preferentially occur in the cytoplasmic segment

A systematic survey of intrinsically disordered (ID) regions was carried out in 2109 human plasma membrane proteins with full assignment of the transmembrane topology with respect to the lipid bilayer. ID regions with 30 consecutive residues or more were detected in 41.0% of the human proteins, a much higher percentage than the corresponding figure (4.7%) for inner membrane proteins of Escherichia coli. The domain organization of each of the membrane protein in terms of transmembrane helices, structural domains, ID, and unassigned regions as well as the distinction of inside or outside of the cell was determined. Long ID regions constitute 13.3 and 3.5% of the human plasma membrane proteins on the inside and outside of the cell, respectively, showing that they preferentially occur on the cytoplasmic side. We interpret this phenomenon as a reflection of the general scarcity of ID regions on the extracellular side and their relative abundance on the cytoplasmic side in multicellular eukaryotic organisms.