scholarly journals Changes in the quaternary structure and function of MjHSP16.5 attributable to deletion of the IXI motif and introduction of the substitution, R107G, in the α -crystallin domain

2013 ◽  
Vol 368 (1617) ◽  
pp. 20120327 ◽  
Author(s):  
Roy A. Quinlan ◽  
Yan Zhang ◽  
Andrew Lansbury ◽  
Ian Williamson ◽  
Ehmke Pohl ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Room Temperature ◽  
Quaternary Structure ◽  
Citrate Synthase ◽  
Small Heat Shock Protein ◽  
Octahedral Symmetry ◽  
Functional Consequences ◽  
And Function ◽  
Induced Aggregation

The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the in vitro chaperone activity with citrate synthase as the client protein. Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer–dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting β L -crystallin against heat-induced aggregation.

scholarly journals Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity

2004 ◽  
Vol 381 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Tirumala Kumar CHOWDARY ◽  
Bakthisaran RAMAN ◽  
Tangirala RAMAKRISHNA ◽  
Chintalagiri Mohan RAO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Quaternary Structure ◽  
Citrate Synthase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Nucleotide Sequence Analysis ◽  
Heat Shock Factor 1 ◽  
Induced Aggregation

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

ArHsp21, a developmentally regulated small heat-shock protein synthesized in diapausing embryos of Artemia franciscana

2008 ◽  
Vol 411 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Zhijun Qiu ◽  
Thomas H. MacRae
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Citrate Synthase ◽  
Sequence Similarity ◽  
Single Gene ◽  
Small Heat Shock Protein ◽  
Subtractive Hybridization ◽  
Artemia Franciscana ◽  
Induced Aggregation

Embryos of the crustacean, Artemia franciscana, undergo alternative developmental pathways, producing either larvae or encysted embryos (cysts). The cysts enter diapause, characterized by exceptionally high resistance to environmental stress, a condition thought to involve the sHSP (small heat-shock protein), p26. Subtractive hybridization has revealed another sHSP, termed ArHsp21, in diapause-destined Artemia embryos. ArHsp21 shares sequence similarity with p26 and sHSPs from other organisms, especially in the α-crystallin domain. ArHsp21 is the product of a single gene and its synthesis occurred exclusively in diapause-destined embryos. Specifically, ArHsp21 mRNA appeared 2 days post-fertilization, followed 1 day later by the protein, and then increased until embryo release at day 5. No ArHsp21 protein was detected in embryos developing directly into larvae, although there was a small amount of mRNA at 3 days post-fertilization. The protein was degraded during post-diapause development and had disappeared completely from second instar larvae. ArHsp21 formed large oligomers in encysted embryos and transformed bacteria. When purified from bacteria, ArHsp21 functioned as a molecular chaperone in vitro, preventing heat-induced aggregation of citrate synthase and reduction-driven denaturation of insulin. Sequence characteristics, synthesis patterns and functional properties demonstrate clearly that ArHsp21 is an sHSP able to chaperone other proteins and contribute to stress tolerance during diapause. As such, ArHsp21 would augment p26 chaperone activity and it may also possess novel activities that benefit Artemia embryos exposed to stress.

scholarly journals Storage temperature determines platelet GPVI levels and function in mice and humans

2021 ◽  
Author(s):  
Jeffrey Miles ◽  
S. Lawrence Bailey ◽  
Ava M Obenaus ◽  
Molly Y Mollica ◽  
Chomkan Usaneerungrueng ◽  
...  
Keyword(s):  
Cold Storage ◽  
Cold Exposure ◽  
Room Temperature ◽  
Storage Temperature ◽  
Storage Duration ◽  
Functional Consequences ◽  
And Function ◽  
Contractile Forces

Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) and room temperature-stored platelets (RSP) in vitro, in vivo, and post-transfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared to RSP. We found significantly reduced GPVI levels after cold exposure of 5-7 days. After transfusion in humans, CSP were mostly equivalent to RSP yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of CSP and highlight the need for thorough investigations of this product. (NCT03787927)

scholarly journals Small heat shock protein of Methanococcus jannaschii, a hyperthermophile

1998 ◽  
Vol 95 (16) ◽  
pp. 9129-9133 ◽  
Author(s):  
Rosalind Kim ◽  
Kyeong Kyu Kim ◽  
Hisao Yokota ◽  
Sung-Hou Kim
Keyword(s):  
Heat Shock ◽  
Citrate Synthase ◽  
Thermal Protection ◽  
Small Heat Shock Protein ◽  
Methanococcus Jannaschii ◽  
Single Chain ◽  
Cell Extracts ◽  
Electron Microscopy Analysis ◽  
Shock Proteins

Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDa proteins that are ubiquitous and are conserved in amino acid sequence among all organisms. A sHSP homologue of Methanococcus jannaschii, a hyperthermophilic Archaeon, forms a homogeneous multimer comprised of 24 monomers with a molecular mass of 400 kDa in contrast to other sHSPs that show heterogeneous oligomeric complexes. Electron microscopy analysis revealed a spherically shaped oligomeric structure ≈15–20 nm in diameter. The protein confers thermal protection of other proteins in vitro as found in other sHSPs. Escherichia coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C. Similar results were obtained when purified sHSP protein was added to an E. coli cell lysate. The protein also prevented the aggregation of two purified proteins: single-chain monellin (SCM) at 80°C and citrate synthase at 40°C.

scholarly journals The effect of in vitro exposure to antisense oligonucleotides on macrophage morphology and function

2011 ◽  
Vol 2 (1) ◽  
pp. 12 ◽  
Author(s):  
Ann Brasey ◽  
Raouf Igue ◽  
Loubna Djemame ◽  
Serge Séguin ◽  
Paolo Renzi ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Morphological Changes ◽  
Lower Airway ◽  
Functional Responses ◽  
Phagocytic Capacity ◽  
Proinflammatory Mediators ◽  
Functional Consequences ◽  
And Function

<p>Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a <em>foamy</em> appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized <em>in vitro</em> using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1<em>α </em>and TNF<em>α</em>, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON<em> in vitro</em>, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the <em>in vivo </em>phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages.</p>

scholarly journals Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

2003 ◽  
Vol 185 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Yinghua Chen ◽  
Catherine Birck ◽  
Jean-Pierre Samama ◽  
F. Marion Hulett
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Salt Bridge ◽  
Pho Regulon ◽  
Dimer Interface ◽  
And Function ◽  
Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

scholarly journals Structure-function relationships in mitochondrial transcriptional condensates

2022 ◽  
Author(s):  
Marina Feric ◽  
Azadeh Sarfallah ◽  
Furqan Dar ◽  
Dmitry Temiakov ◽  
Rohit V Pappu ◽  
...  
Keyword(s):  
Structure Function ◽  
Computational Modelling ◽  
In Vivo Studies ◽  
Bulk Solution ◽  
The Core ◽  
Functional Consequences ◽  
Cellular Machinery ◽  
And Function

Phase separation organizes many membraneless structures in cells. The functional consequences of concentrating cellular machinery into biomolecular condensates, however, is largely unclear. Here, we use in vitro reconstitutions, in vivo studies, and computational modelling to uncover structure-function relationships of mitochondrial (mt-) transcriptional condensates. In vitro, we find that the core mt-transcription machinery — consisting of POLRMT, TFAM, TFB2M, and DNA — forms viscoelastic, multi-phasic condensates. Strikingly, the rates of condensate-mediated transcription are considerably lower than equivalent reactions in bulk solution. Dampened rates are associated with reduced diffusivities of components that become kinetically arrested in non-equilibrium, vesicular condensates. Perturbation of mt-components in vivo and computational simulations recapitulate the transcription-dependent reorganizations observed in vitro. Our findings demonstrate close, bidirectional interdependence between structure and function of transcriptional condensates.

scholarly journals Diselenide Crosslinks for Enhanced and Simplified Oxidative Protein Folding

2020 ◽  
Author(s):  
Reem Mousa ◽  
Taghreed Hidmi ◽  
Sergei Pomyalov ◽  
Shifra Lansky ◽  
Lareen Khouri ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Crystal Structure Analysis ◽  
Folding Rate ◽  
Oxidative Folding ◽  
Functional Studies ◽  
Oxidative Protein Folding ◽  
And Function

<p>The oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. A well-known folding model for many disulfide-rich proteins is that of hirudin. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and folds through plagued pathway that involve highly heterogeneous intermediates and scrambled isomers. The formation of scrambled species is known to limit the rate and efficiency of <i>in vitro</i> oxidative folding of many proteins.</p><p>In the current manuscript we describe our recent work, intended to overcome the limitations of scrambled isomers formation during oxidative protein folding. In this research we deeply investigate the utility of introducing diselenide bridges at the three native disulfide crosslinks as well as at a non-native position on hirudin’s folding, structure and function. Our studies demonstrated that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the hirudin analogs, while reducing the complexity and heterogeneity of the process, and reducing the formation of scrambled isomers.</p><p>A parallel, equally important, objective of our study was to test if diselenide substitutions have structural and functional effects. Crystal structure analysis as well as functional studies indicated that diselenide crosslinks maintained the overall structure of the protein without causing major changes in function and structure. To substantiate these conclusions, we provide inhibition studies and high-resolution crystal structure of the wild-type hirudin and its seleno-analogs. </p>Taken together, we believe that the choice of hirudin as the model in this study has implications beyond its specific folding mechanism, and will serve as a useful methodology for the <i>in vitro</i> oxidative folding of many complex disulfide-rich proteins.

scholarly journals Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

2016 ◽  
Vol 113 (29) ◽  
pp. E4151-E4160 ◽  
Author(s):  
Selom K. Doamekpor ◽  
Joong-Won Lee ◽  
Nathaniel L. Hepowit ◽  
Cheng Wu ◽  
Clement Charenton ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Quality ◽  
E3 Ligase ◽  
Structure And Function ◽  
Dependent Manner ◽  
Ribosomal Subunits ◽  
Terminal Domain ◽  
Cell Extracts ◽  
And Function

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

scholarly journals Crescerin uses a TOG domain array to regulate microtubules in the primary cilium

2015 ◽  
Vol 26 (23) ◽  
pp. 4248-4264 ◽  
Author(s):  
Alakananda Das ◽  
Daniel J. Dickinson ◽  
Cameron C. Wood ◽  
Bob Goldstein ◽  
Kevin C. Slep
Keyword(s):  
Crystal Structure ◽  
Binding Activity ◽  
Structure And Function ◽  
Sensory Disorders ◽  
Tubulin Binding ◽  
Sensory Cilia ◽  
And Function ◽  
Binding Determinants ◽  
Extracellular Environment

Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.

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