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Results for “Intrinsically Disordered Proteins”

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PubMed2006

Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders.

Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.

Heat-Shock Proteins↗

PubMed1998

Genetic defects of folate and cobalamin metabolism.

Deficient activity of an enzyme can result from a defect in the conversion of the vitamin to a co-enzyme as well from an abnormal apo-enzyme or disturbed binding of coenzyme to enzyme. Conversion of dietary vitamin to intracellular active co-enzyme can be complex and require many physiological and biochemical processes including stomach release of bound vitamin, intestinal uptake, carriers/transport, blood transport, cellular uptake, intracellular release and intracellular compartmentalisation. Disorders of malabsorption (food cobalamin malabsorption, intrinsic factor deficiency and abnormal enterocyte cobalamin processing) and transport proteins (transcobalamin II deficiency, R-binder deficiency) mostly lead to disturbed function of the two cobalamin requiring enzymes, methylmalonyl CoA mutase and methionine synthase. Defects of early steps of intracellular cobalamin (cblF, cbl C/D) result in marked deficiencies of both cobalamin co-enzymes and homocystinuria combined with methylmalonic aciduria. Defective synthesis of adenosyl cobalamin in the cbl A/B defects leads to methylmalonyl CoA mutase. Isolated methionine synthase deficiency is also classified as a cobalamin disorder due to its associated deficient formation of methylcobalamin. Folate disorders include methylene-tetrahydrofolate reductase deficiency and glutamate formimino-transferase deficiency. In addition a hereditary disorder of intestinal folate transport has been described. Less well established are disorders of dihydrofolate reductase, methenyl-tetrahydrofolate cyclohydrolase, and defects of cellular folate uptake.

Absorption↗

PubMed2005

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded.

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP' part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

Amino Acid Sequence↗

PubMed2000

Intrinsic structural disorder of the C-terminal activation domain from the bZIP transcription factor Fos.

The bZIP proto-oncoprotein c-Fos activates transcription of a wide variety of genes involved in cell growth. The C-terminal activation domain of c-Fos is functionally independent of the remainder of the protein. Fos-AD corresponds to the C-terminal activation domain of human c-Fos (residues 216-380). Fos-AD suppresses (squelches) transcription in vitro, as expected for a functional activation domain lacking a DNA-binding domain. Fos-AD is unstructured and highly mobile, as demonstrated by circular dichroism spectra indicative of unfolded proteins, a lack of (1)H chemical shift dispersion, and negative (1)H-(15)N heteronuclear nuclear Overhauser effects. The hydrodynamic properties of Fos-AD are also consistent with an extended structure. We conclude that the C-terminal domain of human c-Fos is biologically active yet intrinsically disordered. Our results suggest that conformational disorder is an integral aspect of the diverse contributions to transcriptional regulation by c-Fos.

Basic-Leucine Zipper Transcription Factors↗

PubMed1996

Cloning and characterization of the thyroid iodide transporter.

Iodide (I-) is an essential constituent of the thyroid hormones T3 and T4, and is accumulated by the thyroid. The transport of iodide, the first step in thyroid hormogenesis, is catalysed by the Na+/I- symporter, an intrinsic membrane protein that is crucial for the evaluation, diagnosis and treatment of thyroid disorders. Although several other important thyroid proteins involved in hormogenesis have been characterized, the Na+/I- symporter has not. Here we report the isolation of a complementary DNA clone that encodes this symporter, as a result of functional screening of a cDNA library from a rat thyroid-derived cell line (FRTL-5) in Xenopus laevis oocytes. Oocyte microinjection of an RNA transcript made in vitro from this cDNA clone elicited a more than 700-fold increase in perchlorate-sensitive Na+/I- symport activity over background. To our knowledge, this is the first iodide-transporting molecule to have its cDNA cloned, providing a missing link in the thyroid hormone biosynthetic pathway.

Amino Acid Sequence↗

PubMed2005

Using Drosophila to decipher how mutations associated with human branchio-oto-renal syndrome and optical defects compromise the protein tyrosine phosphatase and transcriptional functions of eyes absent.

Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR- and OD-associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD-associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contribute to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.

Alleles↗

PubMed2026

Auxin-induced ARF transcription factor degradation defines tissue boundaries.

How organs partition themselves into discrete domains with distinct functions is a fundamental question in biology. The gynoecium of flowering plants provides an excellent system to address this question. Here, we show that the boundary between the stigma and style at the gynoecium apex is established by the complementary distribution of the phytohormone auxin and the Auxin Response Factor (ARF), ETTIN (ETT). Mechanistically, auxin induces ETT protein destabilization via the ubiquitin-proteasome pathway. A short sequence motif within an intrinsically disordered region is required for this auxin-triggered degradation. Disruption of this motif leads to ectopic ETT accumulation at the gynoecium apex and consequently abolishes stigma-style boundary development. We further demonstrate that this previously unrecognized mode of auxin-induced ARF instability is evolutionarily conserved among ETT orthologs across angiosperms. In summary, this study reveals how graded auxin distribution affects ARF transcription factor activity, contributing to the establishment of the stigma-style boundary, ensuring correct gynoecium formation and reproductive success in flowering plants.

Indoleacetic Acids↗

PubMed1978

Newer aspects of pernicious anemia.

Although readily treatable with vitamin B12, pernicious anemia continues to captivate investigative endeavors of those interested in the pathophysiology and pathogenesis of this disorder. Notable advances have been made in understanding properties of intrinsic factor, vitamin B12-binding proteins, structure and de novo synthesis of vitamin B12, mechanism of action of vitamin B12-dependent enzymes in man, and metabolic consequences of reduced activities of these enzymes in pernicious anemia. Similarly, newer morphological observations have given information regarding pathogenesis of some of the cytological abnormalities found in megaloblasts, and recent cytochemical studies have shed light on abnormalities of nuclear and cytoplasmic constituents in vitamin B12-deficient cells. Both cellular and humoral factors may contribute to immune-mediated processes in pernicious anemia, although as yet, it has not been established with certainty that pernicious anemia is an autoimmune disorder. As we look ahead, it will be important to define the process or processes responsible for atrophic gastritis, which is the pathophysiological basis of pernicious anemia. Likewise, advances in biophysics used in the study of cell membranes, cell surface phenomena, and metallic ion transport may find applicability in the study of pernicious anemia and perhaps provide further insights into metabolic abnormalities responsible for the development of megaloblastosis.

Anemia, Pernicious↗

PubMed2006

Extrinsic interactions dominate helical propensity in coupled binding and folding of the lactose repressor protein hinge helix.

A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short alpha-helices when bound to DNA but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge-helix folding and/or hinge-hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality.

Bacterial Proteins↗

PubMed2003

HMGA proteins: flexibility finds a nuclear niche?

The mammalian HMGA family of chromatin proteins possesses an unusual constellation of physical, biochemical, and biological characteristics that distinguish them from other nuclear proteins. Principal among these is the fact that, unlike other proteins, they possess little detectable secondary structure prior to interactions with other macromolecules (DNA, RNA, proteins). Upon binding to substrates, however, the HMGA proteins undergo specific disordered-to-ordered structural transitions and also induce alterations in the structure of the substrates themselves. Their intrinsic structural flexibility, combined with other features such as the control of their substrate interactions via complex patterns of in vivo biochemical modifications, allows the HMGA proteins to actively participate in a wide variety of nuclear activities including DNA replication, DNA repair, chromatin remodeling, control of gene transcription, and regulation of mRNA processing

Amino Acid Sequence↗

PubMed2006

Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences.

In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.

Amino Acid Sequence↗

PubMed2006

Folding of the repeat domain of tau upon binding to lipid surfaces.

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.

Amino Acid Sequence↗

PubMed2007

pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model.

BACKGROUND: Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. RESULTS: Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0). The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. CONCLUSION: The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of appoximately 96% at a specificity of approximately 94%). The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. AVAILABILITY: The supplementary data as well as the prediction tool as WWW server are available at http://mendel.imp.univie.ac.at/sat/pkaPS. REVIEWERS: Erik van Nimwegen (Biozentrum, University of Basel, Switzerland), Sandor Pongor (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Igor Zhulin (University of Tennessee, Oak Ridge National Laboratory, USA).

Journal Article↗

PubMed2026

The pseudokinase domain PK1 of UNC-89/obscurin is required for mitochondrial morphology and function in C. elegans.

UNC-89 is a giant modular protein located at the sarcomeric M-line of C. elegans striated muscle and is required for sarcomere organization and function. UNC-89 contains two protein kinase domains, PK1 and PK2, separated by 850 residues, that includes a 645-residue long intrinsically disordered sequence that acts like an elastic spring. Bioinformatic analysis suggests that PK2 is an active kinase whereas PK1 is a pseudokinase. We recently reported that a genome-edited worm, unc-89(sf22), that expresses UNC-89 carrying a kinase-inactivating point mutation in PK2 has an unusual phenotype with normally organized sarcomeres and SR, normal muscle function and yet fragmented mitochondria, increased ATP levels, increased glycolysis and alterations in electron transport chain complexes and respiration. Here, we show that a genome-edited worm unc-89(sf23), that expresses UNC-89 with an in-frame deletion of the C-lobe of PK1 has approximately the same phenotype as the PK2 catalytically dead mutant. The fact that mutations in two different regions of UNC-89 result in a mitochondrial phenotype is further evidence of communication between the sarcomere and mitochondria. We further demonstrate that in vitro PK2 interacts with full length PK1 and the C-lobe of PK1. The protein kinase domains of giant sarcomeric proteins are autoinhibited by parts of their own sequence, and this is also likely for PK2, but the mechanism by which PK2 would be activated is unknown. Our data is compatible with a model in which PK1 interacts with PK2 and thereby stimulates PK2 kinase activity.

Animals↗

PubMed2007

Purification and spectroscopic characterization of the recombinant BG21 isoform of murine golli myelin basic protein.

A recombinant form of the murine Golli-myelin basic protein (MBP) isoform BG21 (rmBG21) has been expressed in E. coli, and isolated to 96% purity via metal chelation chromatography. Characteristic yields were 6-8 mg protein per liter of culture in either minimal M9 or standard Luria-Bertani media. Circular dichroism spectroscopy showed that rmBG21 had a large proportion of random coil in aqueous solution, but gained alpha-helix in the presence of monosialoganglioside G(M1) and PI(4)P, as well as in the membrane-mimetic solvent trifluoroethanol. Bioinformatics analyses of the amino acid sequence of rmBG21 predicted an N-terminal calmodulin (CaM)-binding site. It was determined by fluorescence spectroscopy and dynamic light scattering that rmBG21 and CaM interacted weakly in a 1:1 ratio in a Ca(2+)-dependent manner. Solution NMR spectra of uniformly [(13)C(15)N]-labeled protein in aqueous buffer were consistent with it being an extended protein; spectral quality was independent of temperature. Thus, like "classic" MBP and the Golli-MBP isoform J37, rmBG21 is intrinsically disordered, implying multi functionality, and that its conformation depends on its environment and bound ligands.

Amino Acid Sequence↗

PubMed2002

Functional consequences of preorganized helical structure in the intrinsically disordered cell-cycle inhibitor p27(Kip1).

p27(Kip1) contributes to cell-cycle regulation by inhibiting cyclin-dependent kinase (Cdk) activity. The p27 Cdk-inhibition domain has an ordered conformation comprising an alpha-helix, a 3(10) helix, and beta-structure when bound to cyclin A-Cdk2. In contrast, the unbound p27 Cdk-inhibition domain is intrinsically disordered (natively unfolded) as shown by circular dichroism spectroscopy, lack of chemical-shift dispersion, and negative heteronuclear nuclear Overhauser effects. The intrinsic disorder is not due to the excision of the Cdk-inhibition domain from p27, since circular dichroism spectra of the full-length protein are also indicative of a largely unfolded protein. Both the inhibition domain and full-length p27 are active as cyclin A-Cdk2 inhibitors. Using circular dichroism and proline mutagenesis, we demonstrate that the unbound p27 Cdk-inhibition domain is not completely unfolded. The domain contains marginally stable helical structure that presages the alpha-helix, but not the 3(10) helix, adopted upon binding cyclin A-Cdk2. Increasing or reducing the stability of the partially preformed alpha-helix in the isolated p27 domain with alanine or proline substitutions did not affect formation of the p27-inhibited cyclin A-Cdk2 complex in energetic terms. However, stabilization of the helix with alanine hindered kinetically the formation of the inhibited complex, suggesting that p27 derives a kinetic advantage from intrinsic structural disorder.

Amino Acid Substitution↗

PubMed2003

A protein-chameleon: conformational plasticity of alpha-synuclein, a disordered protein involved in neurodegenerative disorders.

Under the physiological conditions in vitro, alpha-synuclein, a conservative presynaptic protein, the aggregation and fibrillation of which is assumed to be involved into the pathogenesis of Parkinson's disease and several other neurodegenerative disorders, known as synucleinopathies, is characterized by the lack of rigid well-defined structure; i.e., it belongs to the class of intrinsically unstructured proteins. Intriguingly, alpha-synuclein is characterized by a remarkable conformational plasticity, adopting a series of different conformations depending on the environment. For example, this protein may either stay substantially unfolded, or adopt an amyloidogenic partially folded conformation, or fold into alpha-helical or beta-structural species, both monomeric and oligomeric. Furthermore, it might form several morphologically different types of aggregates, including oligomers (spheres or doughnuts), amorphous aggregates, and or amyloid-like fibrils. The peculiarities of this astonishing conformational behavior are analyzed to shed light on structural plasticity of this protein-chameleon.

Alcohols↗

PubMed1998

Intrinsic dendritic cell abnormalities in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immune deficiency, eczema, and microthrombocytopenia. Biochemical evidence indicates that the Wiskott-Aldrich syndrome protein (WASp) is involved in regulating the actin cytoskeleton. Here we report that WAS dendritic cells (DC) have an immunophenotype very similar to normal DC. However, as a consequence of an intrinsically abnormal cytoarchitecture, they are unable to polarize normally and have severely reduced translocational motility in vitro. These findings indicate that WASp is an essential effector for Cdc-42-mediated polarization of primary hematopoietic cells, and suggest that a significant component of the clinical phenotype of WAS could arise from peripheral DC dysmotility and aberrant immune cell trafficking in vivo. Intrinsic dysfunction of the DC population may also have an important role in the pathogenesis of other primary immunodeficiency syndromes, while induced changes in DC cytoskeletal signaling pathways may contribute to the initiation of acquired immunological and inflammatory disorders.

Actins↗