Bacterial toxins induce heat shock proteins in human neutrophils

We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polymorphonuclear granulocytes (PMNs). As was shown by Western blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml) no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as was determined by PCR-analysis.     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

With progressing recognition of apoptosis in bio-logical and medical sciences, the apoptotic signal transduction has rapidly become a dominant project to reveal the molecular mechanisms of apoptotic process. A lot of researches about apoptotic signal transduction have showed the expression of heat shock proteins was closely correlated with cell growth and differen-tiation, and involved in the regulation of apoptosis in different signal transduction pathways. Here we re-view the effects of hsps…     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

Heterooligomeric complexes of human small heat shock proteins

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.     

Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria

Mycobacterium tuberculosis (Mtb) survive inside macrophages by manipulating microbicidal functions such as phago-lysosome fusion, production of reactive oxygen species and nitric oxide, and by rendering macrophages non-responsive to IFN-gamma. Mtb-infected lung tissue does however not only contain macrophages, but also significant numbers of infiltrating polymorphonuclear neutrophils (PMN). These are able to phagocytose and kill ingested Mtb, but are short-lived cells that constantly need to be removed from tissues to avoid tissue damage. Phagocytosis of aged or UV-induced apoptotic PMN by macrophages induce an anti-inflammatory response in macrophages. However, in the present study, we show that engulfment of Mtb-induced apoptotic PMN by macrophages initiates secretion of TNF-alpha from the macrophages, reflecting a pro-inflammatory response. Moreover, Mtb-induced apoptotic PMN up-regulate heat shock proteins 60 and 72 (Hsp60, Hsp72) intracellularly and also release Hsp72 extracellularly. We found that both recombinant Hsp72 and released Hsp72 enhanced the pro-inflammatory response to both Mtb-induced apoptotic PMN and Mtb. This stimulatory effect of the supernatant was abrogated by depleting the Hsp72 with immunoprecipitation. These findings indicate that released Hsp72 from Mtb-infected PMN can trigger macrophage activation during the early stage of Mtb infections, thereby creating a link between innate and adaptive immunity.     

Barcoding heat shock proteins to human diseases: looking beyond the heat shock response

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR) – and thus generally restoring the disturbed protein homeostasis associated with such diseases – has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or ‘barcoded’ by a different set of HSPs that can rescue specific types of aggregation. Some of these ‘non-canonical’ HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated – so-called chaperonopathies – which are also discussed in this Review.KEY WORDS: Chaperonopathies, Heat shock protein, Protein-aggregation diseases     

Mutations of small heat shock proteins and human congenital diseases

The structure and properties of different members of a large family of small heat shock proteins (sHsp) playing an important role in cell homeostasis are described. Participation of the N-terminal domain in formation of large oligomers and chaperone activity of sHsp is analyzed. The structure of the α-crystallin domain of sHsp is characterized and the role of this domain in sHsp dimerization and chaperone activity is discussed. The properties of the C-terminal region of sHsp are described, and its participation in formation of large oligomers and chaperone activity are analyzed. The data from the literature on HspB1 and HspB3 mutations are presented, and involvement of these mutations in development of certain neurodegenerative diseases is discussed. Mutations of HspB4 are described and data on involvement of these mutations in development of cataract are presented. Multiple effects of HspB5 mutations are analyzed, and data are presented indicating that mutations of this protein are accompanied by development of different congenital diseases, such as cataract and different types of myopathies. The data on HspB6 and HspB8 mutations are presented, and feasible effects of these mutations on proteins structure are analyzed. Probable mechanisms underlying sHsp mutation-induced development of different congenital diseases are discussed.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Actin-associated proteins in human neutrophils: identification and reorganization upon cell activation

The neutrophil cytoskeleton, especially the actin network, is thought to play a crucial role in neutrophil migration. However, little is known on the modulation of this network by actin-associated proteins. We have demonstrated the presence of immuno-reactive forms of alpha-actinin (an actin cross-linking and bundling protein) and vinculin (a putative actin-membrane linker) in human neutrophils using specific antibodies to chicken gizzard vinculin and bovine epithelial alpha-actinin. In contrast, talin, another putative actin-membrane linker protein, could not be detected in significant amounts in human neutrophils using a polyclonal antibody raised against chicken gizzard talin, which reacted with human platelet and lymphocyte talin. We have also analyzed the vinculin and alpha-actinin content of Triton X-100 insoluble cytoskeletons, isolated from resting and activated neutrophils. A small amount of alpha-actinin was already associated with the cytoskeleton of resting cells. Addition of chemotactic peptide to the cells rapidly increased the alpha-actinin content of the cytoskeletons 1.6 to 7-fold. This rapid increase was followed by a slower decrease to a lower level which, after 30 min of stimulation, was still significantly higher than that of control cells. The time-course of the association of alpha-actinin with the cytoskeleton paralleled that of actin association. This stimulus-induced rearrangement of cellular alpha-actin may thus play an important role in determining the structure of actin networks in motile neutrophils. Vinculin in contrast could not be detected in significant amounts in the Triton X-100-insoluble neutrophil cytoskeleton, not even after prolonged stimulation of the cells by chemotactic peptide.     

Pressure activation of the chaperone function of small heat shock proteins.

Small heat shock proteins play an important role in the stress response of cells and in several other cellular functions. They possess chaperone-like activity; i.e. they can bind and protect damaged proteins from aggregation and maintain them in a folding-competent state. Two members of this family were investigated in this work: bovine alpha-crystallin and heat shock protein (HSP)16.5 from the thermophilic archaebacteria Methanococcus jannaschii. We reported earlier the enhancement of chaperone potency of alpha-crystallin by high pressure. We now report the completion of the work with results on HSP16.5. The chaperone potency of both proteins can be enhanced significantly by applying high pressure. Evidence by light scattering, Fourier transform infrared (FT-IR) and tryptophan fluorescence experiments show that while the secondary and tertiary structure of these proteins are not influenced by high pressure, their quatemary structure becomes affected: H bonds between subunits are weakened or broken, tryptophan environments become more polar, oligomers dissociate to some extent. We conclude that the oligomeric structure of both proteins is loosened, resulting in stronger dynamics and in more accessible hydrophobic surfaces. These properties lead to increased chaperone potency.     

Chaperone activity of human small heat shock protein-GST fusion proteins

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.     

Guidelines for the nomenclature of the human heat shock proteins

The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), and HSPB (small HSP) as well as for the human chaperonin families HSPD/E (HSP60/HSP10) and CCT (TRiC). The nomenclature is based largely on the more consistent nomenclature assigned by the HUGO Gene Nomenclature Committee and used in the National Center of Biotechnology Information Entrez Gene database for the heat shock genes. In addition to this nomenclature, we provide a list of the human Entrez Gene IDs and the corresponding Entrez Gene IDs for the mouse orthologs.     

Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages

The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (Mphi) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators, such as lipoxins, act as potent regulators (nanomolar range) of the phagocytic clearance of apoptotic cells. In this study, we have investigated the intriguing possibility that apoptotic cells release signals that promote their clearance by phagocytes. We report that conditioned medium from apoptotic human polymorphonuclear neutrophils (PMN), Jurkat T lymphocytes, and human mesangial cells promote phagocytosis of apoptotic PMN by Mphi and THP-1 cells differentiated to a Mphi-like phenotype. This prophagocytic activity appears to be dose dependent, sensitive to the caspase inhibitor zVAD-fmk, and is associated with actin rearrangement and release of TGF-beta1, but not IL-8. The prophagocytic effect can be blocked by the formyl peptide receptor antagonist Boc2, suggesting that the prophagocytic factor(s) may interact with the lipoxin A(4) receptor, FPRL-1. Using nanoelectrospray liquid chromatography mass spectrometry and immunodepletion and immunoneutralization studies, we have ascertained that annexin-1 and peptide derivatives are putative prophagocytic factors released by apoptotic cells that promote phagocytosis of apoptotic PMN by M[phi] and differentiated THP-1 cells. These data highlight the role of annexin-1 and peptide derivatives in promoting the resolution of inflammation and expand on the therapeutic anti-inflammatory potential of annexin-1.     

The small heat shock proteins and their role in human disease

Small heat shock proteins (sHSPs) function as molecular chaperones, preventing stress induced aggregation of partially denatured proteins and promoting their return to native conformations when favorable conditions pertain. Sequence similarity between sHSPs resides predominately in an internal stretch of residues termed the alpha-crystallin domain, a region usually flanked by two extensions. The poorly conserved N-terminal extension influences oligomer construction and chaperone activity, whereas the flexible C-terminal extension stabilizes quaternary structure and enhances protein/substrate complex solubility. sHSP polypeptides assemble into dynamic oligomers which undergo subunit exchange and they bind a wide range of cellular substrates. As molecular chaperones, the sHSPs protect protein structure and activity, thereby preventing disease, but they may contribute to cell malfunction when perturbed. For example, sHSPs prevent cataract in the mammalian lens and guard against ischemic and reperfusion injury due to heart attack and stroke. On the other hand, mutated sHSPs are implicated in diseases such as desmin-related myopathy and they have an uncertain relationship to neurological disorders including Parkinson's and Alzheimer's disease. This review explores the involvement of sHSPs in disease and their potential for therapeutic intervention.