Chaperone Properties Research Articles

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis.

The endoplasmic reticulum (ER) is a cellular organelle where secretory and membrane proteins, as well as lipids, are synthesized and modified. When cells are subjected to ER stress, an adaptive mechanism referred to as the Unfolded Protein Response (UPR) is triggered to allow the cells to restore homeostasis. Evidence has accumulated that the UPR pathways provide specialized and unique roles in diverse development and metabolic processes. The glucose regulated proteins (GRPs) are traditionally regarded as ER proteins with chaperone and calcium binding properties. The GRPs are constitutively expressed at basal levels in all organs, and as stress-inducible ER chaperones, they are major players in protein folding, assembly and degradation. This conventional concept is augmented by recent discoveries that GRPs can be actively translocated to other cellular locations such as the cell surface, where they assume novel functions that regulate signaling, proliferation, apoptosis and immunity. Recent construction and characterization of mouse models where the gene encoding for the UPR components and the GRPs is genetically altered provide new insights on the physiological contribution of these proteins in vivo. This review highlights recent progress towards the understanding of the role of the UPR and two major GRPs (GRP78 and GRP94) in regulating homeostasis of organs arising from the endoderm, mesoderm and ectoderm. GRP78 and GRP94 exhibit shared and unique functions, and in specific organs their depletion elicits adaptive responses with physiological consequences.

Detection of αS2-casein variants in Chinese yak (Bos grunniens) by PCR-SSCP.

Yak (Bos grunniens) is one of the world’s most remarkable domestic animals which thrive in extreme harshness in alpine environment (Wiener et al. 2003). About 90% of the domestic yaks live in the plateau of western China (Wiener et al. 2003; Zhang et al. 2008). The composition of yak’s milk is higher than that of the cow’s milk (Bos taurus) and goat’s milk (Capra hircus) (Wiener et al. 2003; Li et al. 2010). Therefore, in recent years researchers showed more interest in yak’s milk and its products (Prinzenberg et al. 2008; Zhang et al. 2008; Bai et al. 2010; Li et al. 2010; Cui et al. 2012a, b). The αS2-casein (αS2-CN) family constitutes 10% of the casein fraction in bovine milk, and consists of two major and several minor components (Fox 1992; Rasmussen et al. 1992; Farrell et al. 2004). The αS2-CN is one of the three calcium sensitive caseins in milk, and has calcium binding ability along with its high phosphoserine residues content (McSweeney and Fox 2013). αS2-CN possesses molecular chaperone properties under some stress conditions, such as elevated temperature, it stabilizes a variety of unrelated target proteins to prevent their aggregation (Morgan et al. 2005; Treweek et al. 2011). Further, αS2-CN is predicted to yield peptides with defensing like activity and aid the immune system with its antibacterial properties (Farrell et al. 2009). Preparations or proteolytic digests enriched with αS2-CN may have use in value-added or health promoting dairy products (Lopez-Exposito and Recio 2008). αS2-CN is coded by CSN1S2 gene, in the third location of the whole casein cluster, which is located on chromosome 6 of the bovine genome. CSN1S2 gene is 18.5 kb, includes 18 exons and 17 introns (Ferretti et al. 1990). Milk protein variants show a tight correlation with quantitative and qualitative traits of milk, including milk yield, fat yield, protein

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Arabidopsis NAP-related proteins (NRPs) are soluble nuclear proteins immobilized by heat

Nucleosome assembly protein-related proteins (NRPs) are multifunctional proteins having histone chaperone and phosphatase inhibitor properties. Although it is believed that these proteins are nuclear and bind the chromatin, they can be detected in the cytoplasmic but not in the nuclear protein fraction by immunoblotting analysis. It is shown here that under normal conditions, NRPs are nuclear but soluble and leak out of the nuclei during their purification. However, under elevated temperatures (above 42 °C), NRPs display significantly reduced mobility and are retained in the nuclei during purification probably due to binding other immobile macromolecules in the nucleus. Our observations highlight the necessity to use different techniques in parallel to unambiguously determine the intracellular localization of proteins. As heat adapted (38 °C, 2 h followed by 2 h recovery) and heat shocked (45 °C, 1 h), Arabidopsis seedlings were found to have phenotypes similar to those observed in the NRP loss-of-function mutants nrp1-1 nrp2-1 (short, branching roots, increased bleomycin sensitivity), it was also investigated whether the immobilization of NRPs by heat results in disturbed NRP functions. The results indicated, however, that heat affected the investigated traits independent on the presence of NRPs.

Molecular mechanism of the chaperone function of mini-α-crystallin, a 19-residue peptide of human α-crystallin.

α-Crystallin is the archetypical chaperone of the small heat-shock protein family, all members of which contain the so-called “α-crystallin domain” (ACD). This domain and the N- and C-terminal extensions are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human αA-crystallin called mini-αA-crystallin (MAC) shows chaperone properties similar to those of the parent protein. Subsequent studies have confirmed the function of this peptide, but no studies have addressed the mechanistic basis for the chaperone function of MAC. Using human γD-crystallin (HGD), a key substrate protein for parent α-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding but also binds weakly and reversibly to HGD (Kd ≈ 200–700 μM) even when HGD is in the native state. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent α-crystallin. Using nuclear magnetic resonance spectroscopy, we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state is involved in facilitating its binding to both the domain interface and core regions during the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides that affect their chaperone activity and can potentially be manipulated for translational studies.

Small heat-shock proteins: important players in regulating cellular proteostasis.

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.

Abstract 3607: Modulation of neuroblastoma metabolism by an extracellular chaperone SPARC

Abstract Secreted Protein Acidic and Rich in Cysteine (SPARC) is an extracellular matrix protein with diverse biologic functions. We have shown that SPARC is a scavenger chaperone that regulates matrix degradation, remodeling, and repair. We have also demonstrated that SPARC blocks angiogenesis and inhibits the growth of several cancers, including neuroblastoma. In this study we examined how extracellular chaperone SPARC affects neuroblastoma tumor progression. The chaperone properties of SPARC were characterized with cell biology methods and Surface Plasmon Resonance. Neuroblastoma cell lines derived from the high-risk tumors were treated with SPARC. The effect of SPARC on cancer cell metabolism was characterized using the Seahorse analyzer with and without inhibitors of glycolysis and mitochondrial oxidation. Standard cell culture techniques were used to assess the effect of SPARC-induced metabolic changes on neuroblastoma cell proliferation and survival. We demonstrate that SPARC functions as a chaperone, shuttling extracellular ligands between cellular and extracellular tissue compartments. We characterize physical and biological properties of SPARC-mediated ligand transport, and further demonstrate that as a chaperone, SPARC can significantly affect tissue physiology, to the degree of altering cell metabolism. We show that in addition to inhibition of neuroblastoma progression in vivo, SPARC-induced changes in cellular metabolism severely impair neuroblastoma growth and survival in vitro. This novel mechanism of metabolism regulation by an extracellular chaperone SPARC may lead to additional insights into the therapy of neuroblastoma. Citation Format: Alexandre Chlenski, Marija Dobratic, Helen R. Salwen, Mark Applebaum, Susan L. Cohn. Modulation of neuroblastoma metabolism by an extracellular chaperone SPARC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3607. doi:10.1158/1538-7445.AM2014-3607

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

BackgroundThe nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein.ResultsWe have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (−) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity.ConclusionOur data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5′UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC.

The FAM3 superfamily member ILEI ameliorates Alzheimer's disease-like pathology by destabilizing the penultimate amyloid-β precursor.

Accumulation of amyloid-β peptide (Aβ) in the brain underlies the pathogenesis of Alzheimer's disease (AD). Aβ is produced by β- and γ-secretase-mediated sequential proteolysis of amyloid-β precursor protein (APP). Here we identify a secretory protein named interleukin-like epithelial-mesenchymal transition inducer (ILEI, also known as FAM3 superfamily member C) as a negative regulator of Aβ production. ILEI destabilizes the β-secretase-cleaved APP carboxy-terminal fragment, the penultimate precursor of Aβ, by binding to the γ-secretase complex and interfering with its chaperone properties. Notch signalling and γ-secretase activity are not affected by ILEI. We also show neuronal expression of ILEI and its induction by transforming growth factor-β signalling. The level of secreted ILEI is markedly decreased in the brains of AD patients. Transgenic (Tg) overexpression of ILEI significantly reduces the brain Aβ burden and ameliorates the memory deficit in AD model mice. ILEI may be a plausible target for the development of disease-modifying therapies.

Chaperone Potential of Pulicaria undulata Extract in Preventing Aggregation of Stressed Proteins

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei.

The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Neuroinflammation, Heat Shock Protein 27 Phosphorylation, and Cell Cycle: Role and Targeting

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK-2 or MK2) is a downstream substrate of the p38 MAPK responsible for the signaling events influencing inflammation, cell division and differentiation, apoptosis, and cell motility in response to a wide range of extracellular stimuli. After the failure of p38 MAPK inhibitors in clinical trials, MK2 was unveiled as a potential target to regulate inflammatory cytokines' mRNA stability and translation. Recent work suggests that this mechanism may underlie the pathophysiology of brain disorders associated with inflammation. In addition, MK2 is a prominent kinase that phosphorylates heat shock protein 27 (Hsp27), an intensively investigated biomarker of cancer progression. This phosphorylation decreases the chaperone properties of Hsp27, making MK2 an endogenous inhibitor of Hsp27. MK2 is also one of the major players in the signal transduction pathways activated in response to DNA damage. Experimental evidence highlights the role of MK2 in G(2)/M and the mitotic spindle checkpoints, two mechanisms by which MK2 contributes to the maintenance of genomic stability. Thus, MK2 is considered a good molecular target to increase, in combination with chemotherapeutic agents, the sensitivity of treatment, especially in p53-mutated tumors. This review looks at the functions of MK2 in inflammation, Hsp27 regulation, and cell cycle checkpoint control with a focus on brain pathologies. Analysis of MK2 signaling in various disease models and a summary of the data on MK2 inhibitors suggest novel indications for MK2 inhibitors in addition to their mainstream use against peripheral inflammatory disorders.

Is the Nickel-Dependent Urease Complex of Cryptococcus the Pathogen’s Achilles’ Heel?

ABSTRACTThe nitrogen-scavenging enzyme urease has been coopted in a variety of pathogenic organisms as a virulence factor, most notoriously to neutralize stomach acid and establish infection by the gastric pathogen Helicobacter pylori. The opportunistic fungal pathogen Cryptococcus neoformans also utilizes urease as a virulence factor, only in this case to invade the central nervous system (CNS) via the blood-brain barrier and cause life-threatening meningoencephalitis. A recent study [A. Singh, R. Panting, A. Varma, T. Saijo, K. Waldron, A. Jong, P. Ngamskulrungroj, Y. Chan, J. Rutherford, K. Kwon-Chung, mBio 4(3):e00220-13] genetically and biochemically characterizes the accessory proteins required for successful activation of the urease protein complex, including the essential nickel cofactor. The accessory proteins Ure4, Ure6, and Ure7 are all essential for urease function. Ure7 appears to combine the roles of two bacterial accessory proteins: it incorporates both the GTPase activity and nickel chaperone properties of UreE, a bacterial protein whose homolog is missing in the fungi. An accompanying nickel transporter, Nic1, is responsible for most, but not all, nickel uptake into the fungal cell. Mutants of the core urease protein Ure1, accessory protein Ure7, and transporter Nic1 are all attenuated for invasion of the CNS of mice, and urease activity may directly affect integrity of the tight junction of the endothelial cells of the blood-brain barrier, the network of proteins that limits paracellular permeability. This work highlights the potential of urease, its accessory proteins, and nickel transport as potential chemotherapeutic targets.

Chaperone Properties of Pdia3 Participate in Rapid Membrane Actions of 1α,25-Dihydroxyvitamin D3

Protein disulfide isomerase family A, member 3 (Pdia3) mediates many of the plasma membrane (PM)-associated rapid responses to 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3). It is not well understood how Pdia3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1α,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for Pdia3's ER chaperone function, and examined their role in responses to 1α,25(OH)2D3. Pdia3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for Pdia3. Finally, we determined whether palmitoylation and/or myristoylation were required for Pdia3-mediated responses to 1α,25(OH)2D3. Overexpressing the Pdia3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1α,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overexpressing wild-type Pdia3 (Pdia3Ovr cells). Cells overexpressing Pdia3 with K214A and C406S mutations had PKC activity comparable to untreated controls, indicating that the native response to 1α,25(OH)2D3 also was blocked. Overexpressing Pdia3[-KDEL] increased PM localization and augmented baseline PKC, but the stimulatory effect of 1α,25(OH)2D3 was comparable to that seen in wild-type cultures. In contrast, 1α,25(OH)2D3 increased prostaglandin E2 in Pdia3[±KDEL] cells. Although neither palmitoylation nor myristoylation was required for PM association of Pdia3, myristoylation was needed for PKC activation. These data indicate that both the chaperone functional domains and the subcellular location of Pdia3 control rapid membrane responses to 1α,25(OH)2D3.

Two-Color Fluorescent l-Amino Acid Mimic of Tryptophan for Probing Peptide–Nucleic Acid Complexes

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.

Alternative bacterial two-component small heat shock protein systems

Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.

Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1

RNA chaperones are proteins able to rearrange nucleic acid structures towards their most stable conformations. In retroviruses, the reverse transcription of the viral RNA requires multiple and complex nucleic acid rearrangements that need to be chaperoned. HIV-1 has evolved different viral-encoded proteins with chaperone activity, notably Tat and the well described nucleocapsid protein NCp7. We propose here an overview of the recent reports that examine and compare the nucleic acid chaperone properties of Tat and NCp7 during reverse transcription to illustrate the variety of mechanisms of action of the nucleic acid chaperone proteins.