Unfolded Proteins (unfolded + protein)

Distribution by Scientific Domains

Terms modified by Unfolded Proteins

  • unfolded protein response

  • Selected Abstracts


    The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in , -cells

    DIABETES OBESITY & METABOLISM, Issue 2010
    R. J. Kaufman
    Diabetes is an epidemic of worldwide proportions caused by , -cell failure. Nutrient fluctuations and insulin resistance drive , -cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the , -subunit (eIF2,) to attenuate protein synthesis, thereby reducing the biosynthetic burden. , -Cells uniquely require PERK-mediated phosphorylation of eIF2, to preserve cell function. Unabated protein synthesis in , -cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of , -cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases , -cell mass and prevents , -cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes. [source]


    Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin

    PROTEIN SCIENCE, Issue 2 2007
    Franc Avbelj
    Abstract Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants 3J(H,,HN) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating ,-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such ,-strands populate more frequently the left side of the broad , region of ,,, space. Urea-denatured ubiquitin contains no detectable ,-sheet secondary structures; nevertheless, the fluctuating ,-strands in urea-denatured ubiquitin coincide to the ,-strands in the native state. Formation of ,-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of ,-strands in denatured proteins. We propose that fluctuating ,-strands in denatured proteins may serve as initiation sites to form fibrils. [source]


    The kinetics of G-CSF folding

    PROTEIN SCIENCE, Issue 10 2002
    David N. Brems
    Abstract The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by circular dichroism. G-CSF contains two trp residues, and their contribution to the fluorescent-detected kinetics were deciphered through the use of single-site trp mutants. The trp moieties are probes of the local conformation surrounding their environment. One trp at residue 118 is located within the third helix while the other trp at residue 58 is part of the long loop between the first and second helices. The refolding results were most consistent with the following mechanism: U , I1 , I2 , N; where U represents the unfolded protein, I1 represents intermediate state 1, I2 represents intermediate state 2, and N represents the native state. I1 is characterized as having approximately one-half of the native-like helical structure and none of the native-like fluorescence. I2 has 100% of the native helical structure and most of the trp-118 and little of the trp-58 native-like fluorescence. Thus refolding occurs in distinct stages with half of the helix forming first followed by the remaining half of the helix including the third helix and finally the loop between the first and second helices folds. [source]


    Refolding of proteins from inclusion bodies is favored by a diminished hydrophobic effect at elevated pressures

    BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009
    Ryan L. Crisman
    Abstract The application of high hydrostatic pressure is an effective tool to promote dissolution and refolding of protein from aggregates and inclusion bodies while minimizing reaggregation. In this study we explored the mechanism of high-pressure protein refolding by quantitatively assessing the magnitude of the protein,protein interactions both at atmospheric and elevated pressures for T4 lysozyme, in solutions containing various amounts of guanidinium hydrochloride. At atmospheric pressure, the protein, protein interactions are most attractive at moderate guanidinium hydrochloride concentrations (,1,2 molar), as indicated by a minimum in B22 values. In contrast, at a pressure of 1,000 bar no minimum in B22 values is observed, indicating that high pressures colloidally stabilize protein against aggregation. Finally, experimental values of refractive index increments as a function of pressure indicate that at high pressures, wetting of the hydrophobic surfaces is favored, resulting in a reduction of the hydrophobic effect. This reduction in the hydrophobic effect reduces the driving force for aggregation of (partially) unfolded protein. Biotechnol. Bioeng. 2009;102: 483,492. 2008 Wiley Periodicals, Inc. [source]


    Escherichia coli cyclophilin B binds a highly distorted form of trans -prolyl peptide isomer

    FEBS JOURNAL, Issue 18 2004
    Michiko Konno
    Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans -isomer to a cis -isomer in the refolding process of unfolded proteins to recover the natural folding state with cis -proline conformation. To date, only short peptides with a cis -form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans -form proline, i.e. succinyl-Ala- trans -Pro-Ala- p -nitroanilide and acetyl-Ala-Ala- trans -Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis -form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and N,2 of Arg is formed to fix the peptide. On the other hand, in the cis -isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans -isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 N,2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character. [source]


    Autoregulation of the HAC1 gene is required for sustained activation of the yeast unfolded protein response

    GENES TO CELLS, Issue 2 2004
    Naoki Ogawa
    Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating a transcriptional induction program termed the unfolded protein response (UPR). The transcription factor Hac1p responsible for the UPR in Saccharomyces cerevisiae is tightly regulated by a post-transcriptional mechanism. HAC1 mRNA must be spliced in response to ER stress to produce Hac1p, which then activates transcription via direct binding to the cis -acting UPR element (UPRE) present in the promoter regions of its target genes. Here, we show that the HAC1 promoter itself responds to ER stress to induce transcription of its downstream gene, similarly to the KAR2 promoter; the KAR2 gene represents a major target of the UPR. Consistent with this observation, the HAC1 promoter contains an UPRE-like sequence, which is necessary and sufficient for the induction and to which Hac1p binds directly. Cells expressing the HAC1 gene from a mutant HAC1 promoter lacking the HAC1 UPRE could not maintain high levels of either unspliced or spliced HAC1 mRNA and became sensitive to ER stress when insulted for hours. Based on these results, we concluded that autoregulation of the HAC1 genes is required for sustained activation of the UPR and sustained resistance to ER stress. [source]


    Proteasomal degradation of tau protein

    JOURNAL OF NEUROCHEMISTRY, Issue 1 2002
    Della C. David
    Abstract Filamentous inclusions composed of the microtubule-associated protein tau are a defining characteristic of a large number of neurodegenerative diseases. Here we show that tau degradation in stably transfected and non-transfected SH-SY5Y cells is blocked by the irreversible proteasome inhibitor lactacystin. Further, we find that in vitro, natively unfolded tau can be directly processed by the 20S proteasome without a requirement for ubiquitylation, and that a highly reproducible pattern of degradation intermediates is readily detectable during this process. Analysis of these intermediates shows that 20S proteasomal processing of tau is bi-directional, proceeding from both N- and C-termini, and that populations of relatively stable intermediates arise probably because of less efficient digestion of the C-terminal repeat region. Our results are consistent with an in vivo role for the proteasome in tau degradation and support the existence of ubiquitin-independent pathways for the proteasomal degradation of unfolded proteins. [source]


    Ribosome,DnaK interactions in relation to protein folding

    MOLECULAR MICROBIOLOGY, Issue 6 2003
    Jaydip Ghosh
    Summary Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity. [source]


    The hydrodynamic and conformational properties of denatured proteins in dilute solutions

    PROTEIN SCIENCE, Issue 1 2010
    Guy C. Berry
    Abstract Published data on the characterization of unfolded proteins in dilute solutions in aqueous guanidine hydrochloride are analyzed to show that the data are not fit by either the random flight or wormlike chain models for linear chains. The analysis includes data on the intrinsic viscosity, root-mean-square radius of gyration, from small-angle X-ray scattering, and hydrodynamic radius, from the translational diffusion coefficient. It is concluded that residual structure consistent with that deduced from nuclear magnetic resonance on these solutions can explain the dilute solution results in a consistent manner through the presence of ring structures, which otherwise have an essentially flexible coil conformation. The ring structures could be in a state of continual flux and rearrangement. Calculation of the radius of gyration for the random-flight model gives a similar reduction of this measure for chains joined at their endpoints, or those containing loop with two dangling ends, each one-fourth the total length of the chain. This relative insensitivity to the details of the ring structure is taken to support the behavior observed across a range of proteins. [source]


    Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin

    PROTEIN SCIENCE, Issue 2 2007
    Franc Avbelj
    Abstract Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants 3J(H,,HN) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating ,-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such ,-strands populate more frequently the left side of the broad , region of ,,, space. Urea-denatured ubiquitin contains no detectable ,-sheet secondary structures; nevertheless, the fluctuating ,-strands in urea-denatured ubiquitin coincide to the ,-strands in the native state. Formation of ,-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of ,-strands in denatured proteins. We propose that fluctuating ,-strands in denatured proteins may serve as initiation sites to form fibrils. [source]