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.     

Dynamic processes that reflect anti-apoptotic strategies set up by HspB1 (Hsp27)

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis.     

Two Bacterial Small Heat Shock Proteins,IbpA and IbpB,Form a Functional Heterodimer

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other’s expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.     

Microbial small heat shock proteins and their use in biotechnology

Small heat shock proteins (sHsps) exist in almost all organisms. Most organisms have more than one sHsp, but their number can be as high as 65, as found in the eukaryote, Vitis vinifera. The function of sHsps is well-known; they confer thermotolerance to cellular cultures and proteins in cellular extracts during prolonged incubations at elevated temperatures. This demonstrates the ability of sHsps to protect cellular proteins, and to maintain cellular viability under conditions of intensive stress, such as heat shock or chemical treatments. sHsps have several properties that distinguish them from heat shock proteins (Hsps): they function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation, and exhibit a wide range of substrate-binding capacities. Recent studies indicate that sHsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be harnessed for various applications, including nanobiotechnology, proteomics, bioproduction, and bioseparation. In this review, we discuss the properties and diversity of microbial sHsps, as well as their potential uses in the biotechnology industry.     

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.     

Differentially expressed genes in the ovary of the sixth day of pupal “Ming” lethal egg mutant of silkworm, Bombyx mori

The “Ming” lethal egg mutant (l-e^m) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-e^m mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-e^m mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-e^m mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-e^m mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-e^m mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-e^m mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-e^m mutant and Lepidopteran oogenesis in general.     

Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress

The inherent immobility of rice (Oryza sativa L.) limited their abilities to avoid heat stress and required them to contend with heat stress through innate defense abilities in which heat shock proteins played important roles. In this study, Hsp26.7, Hsp23.2, Hsp17.9A, Hsp17.4 and Hsp16.9A were up-regulated in Nipponbare during seedling and anthesis stages in response to heat stress. Subsequently, the expressing levels of these five sHsps in the heat-tolerant rice cultivar, Co39, were all significantly higher than that in the heat-susceptible rice cultivar, Azucena. This indicated that the expressive level of these five sHsps was positively related to the ability of rice plants to avoid heat stress. Thus, the expression level of these five sHsps can be regarded as bio-markers for screening rice cultivars with different abilities to avoid heat stress. Hsp18.1, Hsp17.9A, Hsp17.7 and Hsp16.9A, in the three rice cultivars under heat stress were found to be involved in one protein complex by Native-PAGE, and the interactions of Hsp18.1 and Hsp 17.7, Hsp18.1 and Hsp 17.9A, and Hsp17.7 and Hsp16.9A were further validated by yeast 2-hybridization. Pull down assay also confirmed the interaction between Hsp17.7 and Hsp16.9A in rice under heat stress. In conclusion, the up-regulation of the 5 sHsps is a key step for rice to tolerate heat stress, after that some sHsps assembled into a large hetero-oligomeric complex. In addition, through protein–protein interaction, Hsp101 regulated thiamine biosynthesis, and Hsp82 homology affected nitrogen metabolism, while Hsp81-1 were involved in the maintenance of sugar or starch synthesis in rice plants under heat stress. These results provide new insight into the regulatory mechanism of sHsps in rice.     

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.     

Cellular internalization and stress response of ingested amorphous silica nanoparticles in the midgut of Drosophila melanogaster

Background

Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (< 30 nm) in the midgut of Drosophila melanogaster (Oregon R⁺) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles.

Methods

Third instar larvae of D. melanogaster were exposed orally to 1–100 μg/mL of aSNPs for 12–36 h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints.

Results

A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration.

Conclusion

aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death.

General significance

Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.     

Heat shock proteins: Molecules with assorted functions

Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in all studied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds. Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocation across membranes or mark them for degradation. They are broadly classified in several families according to their molecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play a vital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numerous substrates and are involved in many biological functions such as cellular communication, immune response, protein transport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a brief overview of various Hsps and summarizes their involvement in diverse biological activities.