Small Heat Shock Protein IbpB Acts as a Robust Chaperone in Living Cells by Hierarchically Activating Its Multi-type Substrate-binding Residues

Fu, Xinmiao,Shi, Xiaodong,Yin, Linxiang,Liu, Jiafeng,Joo, Keehyoung,Lee, Jooyoung,Chang, Zengyi American Society for Biochemistry and Molecular Bi 2013 The Journal of biological chemistry Vol.288 No.17

<P>As ubiquitous molecular chaperones, small heat shock proteins (sHSPs) are crucial for protein homeostasis. It is not clear why sHSPs are able to bind a wide spectrum of non-native substrate proteins and how such binding is enhanced by heat shock. Here, by utilizing a genetically incorporated photo-cross-linker (<I>p</I>-benzoyl-<SMALL>l</SMALL>-phenylalanine), we systematically characterized the substrate-binding residues in IbpB (a sHSP from <I>Escherichia coli</I>) in living cells over a wide spectrum of temperatures (from 20 to 50 °C). A total of 20 and 48 residues were identified at normal and heat shock temperatures, respectively. They are not necessarily hydrophobic and can be classified into three types: types I and II were activated at low and normal temperatures, respectively, and type III mediated oligomerization at low temperature but switched to substrate binding at heat shock temperature. In addition, substrate binding of IbpB in living cells began at temperatures as low as 25 °C and was further enhanced upon temperature elevation. Together, these <I>in vivo</I> data provide novel structural insights into the wide substrate spectrum of sHSPs and suggest that sHSP is able to hierarchically activate its multi-type substrate-binding residues and thus act as a robust chaperone in cells under fluctuating growth conditions.</P>

Functional Mode of NtHSP17.6, a Cytosolic Small Heat-Shock Protein from Nicotiana tabacum

( Hae Jeong Yoon ),( Keun Pill Kim ),( Soo Min Park ),( Choo Bong Hong ) 한국식물학회 2005 Journal of Plant Biology Vol.48 No.1

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp, NtHSP17.6, from Nicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation into Escherichia colt and its over-expression, NtHSP17.6 was purified and examined in vitro. This purified NtHSP17.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSP17.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.

A proteomic approach in analyzing heat-responsive proteins in rice leaves

Lee, Dong-Gi,Ahsan, Nagib,Lee, Sang-Hoon,Kang, Kyu Young,Bahk, Jeong Dong,Lee, In-Jung,Lee, Byung-Hyun WILEY-VCH 2007 Proteomics Vol. No.

<P>The present study investigated rice leaf proteome in response to heat stress. Rice seedlings were subjected to a temperature of 42°C and samples were collected 12 and 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress frequently was generated in rice leaves exposed to high temperature. 2-DE, coupled with MS, was used to investigate and identify heat-responsive proteins in rice leaves. In order to identify the low-abundant proteins in leaves, samples were prefractionated by 15% PEG. The PEG supernatant and the pellet fraction samples were separated by 2-DE, and visualized by silver or CBB staining. Approximately 1000 protein spots were reproducibly detected on each gel, wherein 73 protein spots were differentially expressed at least at one time point. Of these differentially expressed proteins, a total of 34 and 39 protein spots were found in the PEG supernatant and pellet fractions, respectively. Using MALDI-TOF MS, a total of 48 proteins were identified. These proteins were categorized into classes related to heat shock proteins, energy and metabolism, redox homeostasis, and regulatory proteins. The results of the present study show that a group of low molecular small heat shock proteins (sHSPs) were newly induced by heat stress. Among these sHSPs, a low molecular weight mitochondrial (Mt) sHSP was validated further by Western blot analysis. Furthermore, four differentially accumulated proteins that correspond to antioxidant enzymes were analyzed at the mRNA level, which confirmed the differential gene expression levels, and revealed that transcription levels were not completely concomitant with translation. The identification of some novel proteins in the heat stress response provides new insights that can lead to a better understanding of the molecular basis of heat-sensitivity in plants.</P>

Biochemical Analysis of a Cytosolic Small Heat Shock Protein, NtHSP18.3, from Nicotiana tabacum

Choo Bong Hong,Ji Hee Yu,Keun Pill Kim,Soo Min Park 한국분자세포생물학회 2005 Molecules and cells Vol.19 No.3

Small heat shock proteins (sHSPs) are widely distributed,and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70°C. Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.

Small Molecule Inhibitors toDisrupt Protein-protein Interactions of Heat Shock Protein 90 Chaperone Machinery

Young Ho Seo 대한암예방학회 2015 Journal of cancer prevention Vol.20 No.1

Heat shock protein 90 (Hsp90) is an adenosine triphosphate dependent molecular chaperone in eukaryotic cells that regulates theactivation and maintenance of numerous regulatory and signaling proteins including epidermal growth factor receptor, human epidermal growth factor receptor 2, mesenchymal-epithelial transition factor, cyclin-dependent kinase-4, protein kinase B, hypoxia-inducible factor 1α, and matrix metalloproteinase-2. Since many of Hsp90 clients are oncogenic proteins, Hsp90 has become an attractive therapeutic target for treatment of cancer. To discover small molecule inhibitors targeting Hsp90 chaperone machinery, several strategies have been employed, which results in three classes of inhibitors such as N-terminal inhibitors, C-terminal inhibitors, and inhibitors disrupting protein-protein interactions of Hsp90 chaperone machinery. Developing small molecule inhibitors that modulate protein-protein interactions of Hsp90 is a challenging task, although it offers many alternative opportunities for therapeutic intervention. The lack of well-definedbinding pocket and starting points for drug design challenges medicinal chemists to discover small molecule inhibitors disrupting protein-protein interactions of Hsp90. The present review will focus on the current studies on small molecule inhibitors disrupting protein-protein interactions of Hsp90 chaperone machinery, provide biological background on the structure, function and mechanism of Hsp90’s protein-protein interactions, and discuss the challenges and promise of its small molecule modulations.

Analysis of the Correlation between Expressions of HSP90α, HSP90β, and GRP94, and the Clinicopathologic Characteristics in Tissues of Non-Small Cell Lung Cancer Patients

김미경,Kim, Mi Kyeong Korean Society for Clinical Laboratory Science 2017 대한임상검사과학회지(KJCLS) Vol.49 No.4

Heat shock proteins (HSPs) are induced as a self-defense mechanism of cells when exposed to various external stresses, such as high fever, infection, free radicals, and heavy metals. They affect the prognosis in the process of tumor formation. HSP is classified into four families: HSP27, HSP60, HSP90, and HSP100, depending on molecular weight. Heat shock protein 90 (HSP90), a molecular chaperone, plays an important role in the cellular protection against various stressful stimuli and in the regulation of cell cycle progression and apoptosis. In the present study, we assessed the differential expression of HSP90 family proteins in non-small cell lung cancer (NSCLC), and the correlation of their expression levels with clinicopathologic factors and patient survival rates. The result of this study can be summarized as follows; $HSP90{\alpha}$ showed higher expression in patients with no lymphovascular invasion (p=0.014). $HSP90{\beta}$ showed a higher expression of squamous cell carcinoma (p=0.003), and an over expression of glucose-related protein (GRP94) was significantly associated with poor differentiation (p=0.048). However, none of the HSP90 proteins showed a significant association with the survival status in patients with NSCLC. This study also indicates that $HSP90{\alpha}$ might contribute more to the carcinogenesis of NSCLC than $HSP90{\beta}$, and GRP94 and isoform selectivity should be considered when HSP90 inhibitors are studied or utilized in the treatment of NSCLC.

Analysis of the Correlation between Expressions of HSP90α, HSP90β, and GRP94, and the Clinicopathologic Characteristics in Tissues of Non-Small Cell Lung Cancer Patients

김미경 대한임상검사과학회 2017 대한임상검사과학회지(KJCLS) Vol.49 No.4

Heat shock proteins (HSPs) are induced as a self-defense mechanism of cells when exposed to various external stresses, such as high fever, infection, free radicals, and heavy metals. They affect the prognosis in the process of tumor formation. HSP is classified into four families: HSP27, HSP60, HSP90, and HSP100, depending on molecular weight. Heat shock protein 90 (HSP90), a molecular chaperone, plays an important role in the cellular protection against various stressful stimuli and in the regulation of cell cycle progression and apoptosis. In the present study, we assessed the differential expression of HSP90 family proteins in non-small cell lung cancer (NSCLC), and the correlation of their expression levels with clinicopathologic factors and patient survival rates. The result of this study can be summarized as follows; HSP90α showed higher expression in patients with no lymphovascular invasion (p=0.014). HSP90β showed a higher expression of squamous cell carcinoma (p=0.003), and an over expression of glucose-related protein (GRP94) was significantly associated with poor differentiation (p=0.048). However, none of the HSP90 proteins showed a significant association with the survival status in patients with NSCLC. This study also indicates that HSP90α might contribute more to the carcinogenesis of NSCLC than HSP90β, and GRP94 and isoform selectivity should be considered when HSP90 inhibitors are studied or utilized in the treatment of NSCLC.

Recent advances in NMR-based structural characterization of αB-crystallin and its potential role in human diseases

Srinivasan Muniyappan,김진해 한국자기공명학회 2019 Journal of the Korean Magnetic Resonance Society Vol.23 No.1

αB-crystallin (αBC) is a member of a small heat-shock protein (sHSP) superfamily and plays a predominant role in cellular protein homeostasis network by rescuing misfolded proteins from irreversible aggregation. αBC assembles into dynamic and polydisperse high molecular weight complexes containing 12 to 48 monomers; this variable stereochemistry of αBC has been linked to quaternary subunit exchange and its chaperone activity. The chaperone activity of αBC poses great potential as therapeutic agents for various neurodegenerative diseases. In this mini-review, we briefly outline the recent advancement in structural characterization of αBCs and its potential role to inhibit protein misfolding and aggregation in various human diseases. In particular, nuclear magnetic resonance (NMR) spectroscopy and its complimentary techniques have contributed much to elucidate highly-dynamic nature of αBCs, among which notable advancements are discussed in detail. We highlight the importance of resolving the structural details of various αBC oligomers, their quaternary dynamics, and structural heterogeneity.

Recent advances in NMR-based structural characterization of αB-crystallin and its potential role in human diseases

Muniyappan, Srinivasan,Kim, Jin Hae Korean Magnetic Resonance Society 2019 Journal of the Korean Magnetic Resonance Society Vol.23 No.1

${\alpha}B$-crystallin (${\alpha}BC$) is a member of a small heat-shock protein (sHSP) superfamily and plays a predominant role in cellular protein homeostasis network by rescuing misfolded proteins from irreversible aggregation. ${\alpha}BC$ assembles into dynamic and polydisperse high molecular weight complexes containing 12 to 48 monomers; this variable stereochemistry of ${\alpha}BC$ has been linked to quaternary subunit exchange and its chaperone activity. The chaperone activity of ${\alpha}BC$ poses great potential as therapeutic agents for various neurodegenerative diseases. In this mini-review, we briefly outline the recent advancement in structural characterization of ${\alpha}BCs$ and its potential role to inhibit protein misfolding and aggregation in various human diseases. In particular, nuclear magnetic resonance (NMR) spectroscopy and its complimentary techniques have contributed much to elucidate highly-dynamic nature of ${\alpha}BCs$, among which notable advancements are discussed in detail. We highlight the importance of resolving the structural details of various ${\alpha}BC$ oligomers, their quaternary dynamics, and structural heterogeneity.

Small heat shock protein genes of the green algae <i>Closterium ehrenbergii</i>: Cloning and differential expression under heat and heavy metal stresses

Abassi, Sofia,Wang, Hui,Ponmani, Thangaraj,Ki, Jang‐,Seu John Wiley Sons, Inc. 2019 Environmental toxicology Vol.34 No.9

<P><B>Abstract</B></P><P>The freshwater green algae <I>Closterium ehrenbergii</I> has been considered as a model for eco‐toxicological assessment in aquatic systems. Heat shock proteins (HSPs) are a class of highly conserved proteins produced in all living organisms, which participate in environmental stress responses. In the present study, we determined the cDNA sequences of small heat shock protein 10 (<I>sHSP10</I>) and <I>sHSP17.1</I> from <I>C. ehrenbergii</I>, and examined the physiological changes and transcriptional responses of the genes after exposure to thermal shock and toxicants treatments. The open reading frame (ORF) of <I>CeHSP10</I> was 300 bp long, encoding 99 amino acid (aa) residues (10.53 kDa) with a GroES chaperonin conserved site of 22 aa. The <I>CeHSP17.1</I> had a 468 bp ORF, encoding 155 aa with a conserved C‐terminal α‐crystallin domain. For heat stress, cells presented pigment loss and possible chloroplast damage, with an up‐regulation in the expression of both <I>sHSP10</I> and <I>sHSP17.1</I> genes. As for the heavy metal stressors, an increase in the production of reactive oxygen species was registered in a dose dependent manner, with a significant up‐regulation of both <I>sHSP10</I> and <I>sHSP17.1</I> genes. These results suggest that <I>sHSP</I> genes in <I>C. ehrenbergii</I> may play a role in responses to stress environments, and they could be used as an early detection parameter as biomarker genes in molecular toxicity assessments.</P>