Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.     

Development and tissue-specific distribution of mouse small heat shock protein hsp25

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13–20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related α, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism. © 1993Wiley-Liss, Inc.     

Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine-rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.     

Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity

During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic alpha-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature,long-term anoxia,desiccation and other wide variety of stressful conditions.How the embryos survive these stresses is a very interesting and unsolved subj...     

关于樟科润楠属和鳄梨属的分类界线问题

讨论了润楠属Machilus Nees与鳄梨属Persea Mill.的分类界线, 认为两属的主要区别如下: 在润楠属中, 花被裂片相等或近相等, 极少外轮明显短于内轮, 在果期为纸质, 极少为薄革质, 伸长, 强度反曲, 极少张开与不反曲, 几乎宿存, 极少脱落, 如果脱落时则内、外轮从基部完全脱落; 花柱早落; 分布亚洲热带及亚热带地区。而在鳄梨属中, 花被裂片极不相等, 外轮明显短于内轮, 很少近相等, 在果期为革质到木质, 很少为薄革质, 张开或直立, 几乎不反曲, 绝大部分宿存或内轮在距基部的1/3到1/2处脱落; 花柱有时宿存; 分布美洲。两属的界线清楚, 不宜合并。把产于越南北部的Persea balansae Airy Shaw和产于苏门答腊的P. sumatrana Kosterm.转移到Machilus中, 从而提出Machilus balansae (Airy Shaw) F. N. Wei &; S. C. Tang和M. sumatrana (Kosterm.) F. N. Wei &; S. C. Tang两个新组合。     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Expression of the small heat shock protein (hsp) 27 in human astrocytomas correlates with histologic grades and tumor growth fractions

Summary 1. Cellular expression and distribution of the stress response small heat shock protein 27 (hsp27) in 39 high-grade astrocytomas (27 glioblastoma multiformes, 12 anaplastic astrocytomas) and in 27 low-grade astrocytomas (grade I–II) were analyzed immunohistochemically.2. The correlation between hsp27 expression and tumor growth fractions of the astrocytomas was examined following Ki-67 immunostaining.3. The hsp27 staining was cell cytoplasmic. The hsp27 immunopositive rate was significantly higher in high-grade astrocytomas; the rates were 74% for glioblastomas, 58% for anaplastic astrocytomas, and 37% for low-grade astrocytomas. The small and large tumor cells, especially in glioblastomas, multinucleated tumor giant cells, tumor cells in the pseudopalisading and necrotic areas, cells of the microvascular endothelial proliferations, and tumor vascular smooth muscles were usually hsp27 positive. The mean percentage of hsp27-positive cells was significantly higher in the glioblastomas alone and in the combined high-grade astrocytomas, compared to the low-grade, and in recurrent rather than in primary high-grade astrocytomas.4. The high-grade astrocytomas had a highly statistical significant Ki-67 labeling index. The Ki-67 labeling indices were significantly higher in the hsp27-positive than the hsp27-negative astrocytomas, irrespective of the histological grade. In the high-grade astrocytomas with a Ki-67 labeling index of five and above, 81% of those tumors were hsp27 positive.5. Thus, a large number of human astrocytomas express hsp27, and hsp27 expression correlates with histological grades of astrocytoma and with tumor growth fractions. This being the case, hsp27 is likely to have a role in the growth of human astrocytomas.     

Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli

Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli.     

Contrasting phylogeographical patterns of two closely related species, Machilus thunbergii and Machilus kusanoi (Lauraceae), in Taiwan

Aim The purpose of this paper was to study the patterns of genetic variation, demographic history, haplotype relationships and potential location of diversity centres of two closely related species, Machilus thunbergii and Machilus kusanoi. Location The phylogeography of M. thunbergii and M. kusanoi was examined by sampling 110 and 106 individuals from 25 and 16 sampling sites, respectively, across their distributional range in Taiwan. Machilus thunbergii is distributed on the Asian mainland, South Korea, southern Japan, the Ryukyus, Taiwan and the Philippines, whereas M. kusanoi is endemic to Taiwan. These two species are closely related, and both are widely distributed in Taiwan but occupy different altitudinal zones and habitats. Methods The range‐wide variation of M. thunbergii and M. kusanoi in Taiwan was studied using chloroplast DNA (cpDNA) variations. A haplotype network was constructed with the computer program tcs . Nested clade analysis was conducted with the computer program ceodis , and various parameters of genetic diversity were calculated and neutrality tested by the computer program Dna SP. Population differentiation was estimated using the programs arlequin and hapstep . The contribution of the populations to gene diversity and to allelic richness was calculated using the software contrib . The level of divergence for each population from the remaining populations was calculated as the mean value of pairwise F_ST for each population against the rest of the populations. Results Extremely low levels of genetic differentiation were found for both species. This result suggested that these two species probably survived in multiple relict refugia with different population sizes throughout the island during low‐temperature periods of the Pleistocene. In addition, nested clade analysis (NCA) of cpDNA haplotypes indicated that restricted gene flow with isolation‐by‐distance characterized the recolonization after the Pleistocene by Tashueshan and Shiouhluan populations of M. thunbergii in the north‐central area west of the Central Mountain Range (CMR). In contrast, NCA analysis indicated that a major diversity centre on the southern tip of the island (Kending population) and contiguous range expansion characterized the recolonization by M. kusanoi of northern areas along the east side of the CMR. The major diversity centres found for the two species examined were further supported by the results of the mean F_ST for individual populations in comparison with other populations, and of the contribution of the divergence component to the total diversity. Main conclusions This research supports the multiple relict refugia hypothesis for both species investigated. Populations of M. thunbergii at Shiouhluan and Tashueshan in the north‐central area west of the CMR represent a diversity centre currently expanding its size. A diversity centre at the southern‐edge population of M. kusanoi, and a contiguous range expansion from Kending, were found. These results indicate that the M. thunbergii populations at Tashueshan and Shiouhluan and the M. kusanoi population at Kending, and even Soukar, are evolutionarily significant units for conservation programmes.     

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs*

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BomIII and BglI.     

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.     

Characterisation of chloroplast heat shock proteins in young leaves of C4 monocotyledons

In vivo radiolabeling of chloroplast proteins in grain sorghum (Sorghum bicolor L. cv. Texas 610) leaves and their separation by one-dimensional electrophoresis revealed at least 6 heat shock proteins (HSPs) between 24 and 94 kDa. of which the 24 kDa protein was the most prominent. All of these chloroplast heat shock proteins were found exclusively in the stroma. The 24 kDa heat shock protein, upon closer examination using two-dimensional electrophoresis proved to be two similarly-sized heat shock polypeptides with identical molecular masses and level of radiolahel incorporation, hut slightly different in isoeiectric points, suggesting isomers. Separation of stromal heat shock proteins synthesised in two other C₄ monocotyledons ( Punicum miliaceum L. and Umchloa panictrides L.) revealed similar putative isomers. each of 24 kDa. Several other, previously unidentified, heat shock proteins between 22 and 38 kDa were also observed in all three species. In P. miliaceum. the most prominent HSP was the pair of 24 kDa proteins, whereas in U. panicoides. it was a group of 35 to 38 kDa HSPs that was most abundant. In vivo chlorophyll fluorescence measurements showed that no sustained impairment to photosynthetic efficiency had occurred for each species after the heat stress regime. However, when cytoplasmic protein synthesis was inhibited during the high temperature treatment, a dramatic decrease was observed in photosynthetic efficiency, suggesting a possible protective role for chloroplast heat shock proteins. It was also shown that a single chloroplast HSP complex of around 380 kDa was observed in the stroma of both 5. bicolor and P. miliaceum leaves in vivo. This was in contrast to the smaller HSP complex (200–265 kDa) observed in previous studies on chloroplast heat shock proteins in C_j species.     

国产润楠属植物的叶表皮特征及其系统学意义

对45种国产润楠属(Machilus Nees)植物的叶表皮进行光学显微镜和扫描电镜观察,其中31种为首次报道.结果表明:润楠属植物叶表皮细胞形状为多边形或不规则形,气孔复合体均为平列型且仅分布在下表皮;上下表皮细胞的垂周壁式样有5种类型:平直、圆滑、浅波、波状、平直-弓形;下表皮毛被有(稀疏、密集或非常密集)或无;上表皮平周壁纹饰类型有:点状、平滑、条纹(或波浪条纹);下表皮内面平周壁纹饰类型有:不规则、平滑、粗糙网状、微毛状、颗粒状、鳞片状.这些特征在本属种间存在较显著的差异,可作为区分种类的辅助特征和种间亲缘关系,因而对润楠属植物的分类学研究和亲缘关系的确定有较大意义.