A Vertical Gradient of the Chloroplast Abundance among Leaves of Chenopodium album

The abundances of chloroplasts in leaves on the main stems ofChenopodium album at different height levels were investigatedin relation to the photosynthetic capacity and light environmentof the leaves. (1) The number of chloroplasts per mesophyllcell decreased with descending position of leaves, except foryoung developing leaves at the top of plants that had smallerchloroplast numbers per cell than matured leaves beneath them.Contents of chlorophyll and ribulose-1,5-bisphosphate carboxylase/oxygenaseper leaf area that were highest in the topmost young leavesand decreased with decreasing height level indicate that thereis a vertical gradient of chloroplast abundance per leaf areadecreasing from the top of the leaf canopy with depth. (2) Light-saturatingrate of photosynthetic oxygen evolution per leaf area of maturedleaves decreased more steeply with decreasing leaf positionthan the chloroplast number per cell. Gradients of chlorophylland the enzyme protein contents were also steeper than thatof the chloroplast number. Loss of photosynthesis in lower leavesis, therefore, ascribed partly to loss of whole chloroplastsand partly to reduced photosynthetic capacities of the remainingchloroplasts. (3) The chloroplast number per cell in newly expandedsecond leaves was comparable to those in leaves that have developedat later stages of the plant growth but decreased graduallyduring leaf senescence both in the dark and light. The formationof the vertical gradient of chloroplast abundance is, therefore,ascribed to loss of whole chloroplasts during senescence ofleaves. (4) Irradiance a leaf receives decreased sharply fromthe top of the canopy with depth. The physiological or ecophysiologicalsignificance of the vertical distribution of chloroplasts amongleaves was discussed taking light environments of leaves intoconsideration. (Received July 31, 1995; Accepted October 20, 1995)     

BOBBER1 Is a Noncanonical Arabidopsis Small Heat Shock Protein Required for Both Development and Thermotolerance

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here, we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis (Arabidopsis thaliana) small heat shock protein. BOB1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss-of-function mutants are embryo lethal, we used a partial loss-of-function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and postembryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants, BOB1 is cytoplasmic at basal temperatures, but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects, bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide, to our knowledge, the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.Plants are autotrophic sessile organisms that depend on sunlight for their energetic needs. One consequence of this lifestyle is that plants are often subjected to high temperature stress, especially in dry conditions when transpirational cooling is limited. At a cellular level, elevated temperatures result in changes in protein structure that can result in the exposure of normally buried hydrophobic residues. As a consequence of thermal denaturation, proteins may aggregate and cease to function normally. A universal response to temperature-induced protein unfolding in all living organisms is the production of heat shock proteins (HSPs). HSPs are molecular chaperones that provide organismal thermotolerance by preventing the denaturation and aggregation of target proteins as well as facilitating protein refolding. Highly conserved HSPs are found in all organisms and include the small HSP (sHSP) as well as the Hsp60, Hsp70, Hsp90, and Hsp100 families (Baniwal et al., 2004; Taiz and Zeiger, 2006). Members of the sHSP family are defined by their small size (12–43 kD), their ability to prevent protein aggregation, and by a conserved α-crystallin domain (ACD). Plants are unusual in the large number of ACD-containing sHSPs encoded by their genomes: Arabidopsis (Arabidopsis thaliana) has 19 compared to 10 in humans, four in Drosophila melanogaster, and one or two in bacteria (Haslbeck et al., 2005).Although the biochemical activity of plant sHSPs has been well characterized (Lee et al., 1995, 1997; Basha et al., 2004; Siddique et al., 2008), little is known about the in vivo functions of plant sHSPs, perhaps due to functional redundancies in this large gene family. Apart from temperature-dependent changes in hypocotyl elongation, which reflects the ability of cells to expand, no developmental roles for a sHSP have been reported in plants (Jenks and Hasegawa, 2005; Dafny-Yelin et al., 2008). In addition to redundancy, a lack of known developmental functions for plant sHSPs may also be a result of the fact that most are only expressed in response to heat or other stresses. Exceptions include a subset of sHSPs expressed during seed and pollen maturation, developmental stages that involve desiccation (Wehmeyer and Vierling, 2000; Dafny-Yelin et al., 2008). However, since most plant sHSPs are not expressed under nonstress conditions, they are unlikely to affect normal growth and development (Swindell et al., 2007).BOBBER1 (BOB1; At5g53400) is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. In bob1-1 and bob1-2 null mutants, meristematic identity is expanded into the portion of the embryo that would normally form the seedling leaves (cotyledons), which in turn are never established. Auxin gradients are never established in bob1 mutant embryos. However, since there are multiple feedback loops involved in auxin signaling and transport, it is unclear whether the lack of auxin maxima in bob1 mutants is a direct or indirect result of a lack of BOB1 activity (Jurkuta et al., 2009). BOB1 encodes a protein with C-terminal homology to NudC, a protein identified in a screen for genes required for nuclear migration in Aspergillus nidulans. Genes with homology to NudC have been shown to interact with dynein microtubule motors. In mammalian tissue culture systems, interference with NudC-like gene function results in defects in chromosome segregation and cytokinesis (Aumais et al., 2003; Nishino et al., 2006; Zhou et al., 2006). The NudC domain has predicted structural homology with the α-crystallin/p23 protein families (Garcia-Ranea et al., 2002), which includes the ACD-containing sHSPs. The ACD, originally identified in the α-crystallin chaperone of the vertebrate eye lens, forms a structure consisting of two antiparallel β-sheets in a sandwich (Scharf et al., 2001; Haslbeck et al., 2005). The NMR structure of the mouse NudC homolog (PDB 1wfi) has the same β-sheet sandwich structure that provides support for the predicted structural homology between NudC domains and ACDs. These observations suggest that NudC domain proteins might share conserved functions with sHSPs. Support for this hypothesis comes from Caenorhabditis elegans where the NudC homolog NUD-1, an essential gene, displays protein chaperone activity in vitro (Faircloth et al., 2009).Here, we use bob1-3, a partial loss-of-function allele, to show that BOB1 is required for normal development and meristem function after embryogenesis. To determine whether BOB1 functions as a protein chaperone, we characterized the in vitro activity of BOB1 protein. We also investigated the thermotolerance functions of BOB1 using bob1-3 and used a BOB1:GFP line that is biologically active to document that BOB1 protein is incorporated into heat shock granules (HSGs) at high temperatures. All of these data suggest that BOB1 encodes a novel sHSP with dual functions in development and thermotolerance. To our knowledge, this is the first demonstration of a developmental patterning function for a plant sHSP.     

A Model for Small Heat Shock Protein Inhibition of Polyglutamine Aggregation

Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protection against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp αB-crystallin, we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.     

Detection and Architecture of Small Heat Shock Protein Monomers

Background

Small Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family.

Methodology/Principal Findings

We developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM).

Conclusions/Significance

This work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers.     

Dissecting the Functional Role of the N-Terminal Domain of the Human Small Heat Shock Protein HSPB6

HSPB6 is a member of the human small heat shock protein (sHSP) family, a conserved group of molecular chaperones that bind partially unfolded proteins and prevent them from aggregating. In vertebrate sHSPs the poorly structured N-terminal domain has been implicated in both chaperone activity and the formation of higher-order oligomers. These two functionally important properties are likely intertwined at the sequence level, complicating attempts to delineate the regions that define them. Differing from the prototypical α-crystallins human HSPB6 has been shown to only form dimers in solution making it more amendable to explore the determinants of chaperoning activity alone. Using a systematic and iterative deletion strategy, we have extensively investigated the role of the N-terminal domain on the chaperone activity of this sHSP. As determined by size-exclusion chromatography and small-angle X-ray scattering, most mutants had a dimeric structure closely resembling that of wild-type HSPB6. The chaperone-like activity was tested using three different substrates, whereby no single truncation, except for complete removal of the N-terminal domain, showed full loss of activity, pointing to the presence of multiple sites for binding unfolding proteins. Intriguingly, we found that the stretch encompassing residues 31 to 35, which is nearly fully conserved across vertebrate sHSPs, acts as a negative regulator of activity, as its deletion greatly enhanced chaperoning capability. Further single point mutational analysis revealed an interplay between the highly conserved residues Q31 and F33 in fine-tuning its function.     

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.     

Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album

Water-soluble chlorophyll (Chl)-binding proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A₇₄₂/A₆₆₈ ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.     

Dynamics and Composition of Small Heat Shock Protein Condensates and Aggregates

Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders.HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures.To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.     

被子植物小热激蛋白家族的比较基因组学分析

小热激蛋白(sHSP)是一类重要的响应外界环境变化以及调控植物生长发育的蛋白家族。基于在睡莲(Nymphaea colorata)、水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和葡萄(Vitis vinifera)中分别鉴定到的33个NcsHSPs、24个OssHSPs、17个A...     

Isolation and Characterization of Two Protein Isoforms with Antiviral Activity from Chenopodium album L Leaves

Two antiviral proteins (AVPs) named CAP-I and CAP-II isolated and purified from the leaves of Chenopodium album cv Pusa Bathua 1 were found to inhibit tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) infection on their respective host plants. The molecular weight of both the AVPs was found to be 24 kD. They were devoid of carbohydrate moiety and were highly basic with pI ～10.2. However, they differed with respect to amino acid composition and N-terminal sequence. They also differed with respect to IC₅₀ values, and CAP-I was found to be 2.5 fold more effective than CAP-II in inhibiting viral infection.     

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments

Biochemistry (Moscow) - Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell...     

A New Role for Escherichia coli DsbC Protein in Protection against Oxidative Stress

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.     

嗜热古菌S.solfataricus小热激蛋白基因的克隆及表达

目的:从嗜热古菌Sulfolobus solfataricus中克隆一种新的小热激蛋白SsHsp14.1的基因,并研究其表达和生物活性。方法:用PCR技术以S.solfataricus基因组为模板扩增得到目的基因序列片段,并将其克隆到pET-28a(+)中,转化到E coli BL21(DE3)中经IPTG诱导表达,纯化后对产物进行生物活性测定。结果:从菌株S.solfataricus中克隆出目的基因,该基因的编码框由375个碱基组成,编码的蛋白质由124个氨基酸组成。含该质粒的大肠杆菌经诱导表达了一个与预期理论值相符的约14kDa的蛋白,利用亲和层析和凝胶柱分离纯化了重组蛋白。试验证明纯化后的重组SsHsp14.1具有分子伴侣活性,重组蛋白在体内表达时能提高E.coli细胞的耐热性。结论:成功克隆SsHsp14.1基因并表达出蛋白,并明确了其分子伴侣活性,为该热激蛋白的研究和应用奠定基础。     

小鼠HSP60基因的克隆与表达条件的优化

目的:克隆小鼠热休克蛋白60(HSP60)基因,在大肠杆菌中表达,并进一步对其表达条件进行优化.方法:采用RTPCR技术克隆出非肥胖性糖尿病(NOD)小鼠HSP60的cDNA序列.构建重组表达载体pET28a- HSP60,以其转化大肠杆菌感受态细胞BL21( DE3),在不同的菌体浓度、不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)、不同诱导时间以及不同温度条件下诱导,检测重组蛋白的表达情况.结果:获得一个含有1 721bp的cDNA的片段,重组蛋白HSP60在E.coli BL -21 (DE3)中的最佳表达条件是菌体密度A600nm为0.6,诱导时间为5h,诱导物(IPTG)浓度为4mmol/L,诱导温度为35℃,重组蛋白大小约为60kD.结论:成功克隆了小鼠HSP60基因,并在大肠杆菌中获得高效表达,为重组蛋白的分离纯化及进一步研究其生物学功能奠定了基础.     

Role for the Cyanobacterial HtpG in Protection from Oxidative Stress

The heat shock protein HtpG, which is a homolog of HSP90, is essential for basal and acquired thermotolerances in cyanobacteria. Recently we demonstrated that HtpG was involved in the acclimation to low temperatures in cyanobacteria. In this study, we elucidated a role of HtpG in the cyanobacterium Synechococcus sp. PCC 7942, in the acclimation to oxidative stress that was caused by high irradiance and/or methyl viologen. The inactivation of the htpG gene resulted in a decrease in the survival rate and an increase in the photoinhibition of photosystem II when cells in a liquid medium were incubated under high light conditions. The htpG mutant was highly sensitive to methyl viologen when it was grown on an agar plate. High irradiance and/or methyl viologen greatly increased the expression of the htpG gene as well as the groEL gene in the wild-type strain. Taken together, our results suggest that HtpG may play a role by itself or with other molecular chaperones in the acclimation to oxidative stress. Received: 1 April 2002 / Accepted: 4 May 2002