Neutrophil proteases degrade autoepitopes of NET-associated proteins.

Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.

Crystal structures of alpha-crystallin domain dimers of alphaB-crystallin and Hsp20.

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

Expression of the small heat shock protein family in the mouse CNS: differential anatomical and biochemical compartmentalization.

The small heat shock proteins (sHsps) are a family of molecular chaperones defined by an alpha-crystallin domain that is important for sHsps oligomerization and chaperone activity. sHsps perform many physiological functions including the maintenance of the cellular cytoskeleton, the regulation of protein aggregation and modulate cell survival in a number of cell types including glial and neuronal cells. Many of these functions have been implicated in disease processes in the CNS and indeed sHsps are considered targets for disease therapy. Despite this, there is no study that systematically and comparatively characterized sHsps expression in the CNS. In the present study we have analyzed the expression of this gene family in the mouse brain by reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting. Gene expression analysis of the 10 known members of mammalian sHsps confirms the presence of 5 sHsps in the CNS. A distinct white matter specific expression pattern for HspB5 and overlapping expression of HspB1 and HspB8 in the lateral and dorsal ventricles of the brain is observed. We confirm protein expression of HspB1, HspB5, HspB6 and HspB8 in the brain. Further subcellular fractionation of brain and synaptosomes details a distinct subcompartment-specific association and detergent solubility of sHsps. This biochemical signature is indicative of an association with synaptic and other neural specializations. This observation will help one understand the functional role played by sHsps during physiology and pathology in the CNS.

Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer's disease brains.

The small heat shock protein family (sHsp) comprises molecular chaperones able to interact with incorrectly folded proteins. Alzheimer's disease (AD) is characterized by pathological lesions such as senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of the incorrectly folded proteins amyloid-beta (Abeta) and tau respectively. The aim of this study was to investigate the association of the chaperones Hsp20, HspB2, alphaB-crystallin and Hsp27 with the pathological lesions of AD brains. For this purpose, a panel of well-characterized antibodies directed against these sHsps was used in immunohistochemistry and immunoblotting. We observed extracellular expression of Hsp20, Hsp27 and HspB2 in classic SPs, and Hsp20 expression in diffuse SPs. In addition, extracellular expression of HspB2 was observed in CAA. Both Hsp27 and alphaB-crystallin were also observed in astrocytes associated with both SPs and CAA. Furthermore, none of the sHsps were observed in NFTs in AD brains. We conclude that specific sHsp species may be involved in the pathogenesis of either SPs or CAA in AD.

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.

Characterization of two novel human small heat shock proteins: protein kinase-related HspB8 and testis-specific HspB9.

Using search profiles based on the conserved alpha-crystallin domain that is characteristic for small heat shock proteins (sHsps), we traced two new human sHsps. One of these, being the eighth known human sHsp and thus named HspB8, was recently described as a serine-threonine protein kinase (H11), but not identified as an sHsp (C.C. Smith, Y.X. Yu, M. Kulka, L. Aurelian, J. Biol. Chem. 275 (2000)). Northern blotting showed that HspB8/H11 is predominantly transcribed in skeletal muscle and heart, like most other sHsps. The other, named HspB9, is specifically expressed in testis, notably in the spermatogenic cells from late pachytene spermatocyte stage till elongate spermatid stage. While mammalian sHsps are generally highly conserved, mouse HspB9 shows 38% sequence difference with human HspB9, which may confirm its sex-related role.

Interaction between alphaB-crystallin and the human 20S proteasomal subunit C8/alpha7.

alphaB-Crystallin, a member of the small heat shock protein (sHsp) family, can bind unfolding proteins, but is unable to refold them. To fulfil its protective function in vivo it is therefore likely to interact with other cellular proteins. Here we report that alphaB-crystallin binds very specifically both in vitro and in vivo to C8/alpha7, one of the 14 subunits of the 20S proteasome. The C8/alpha7 protein forms heterogeneous complexes with alphaB-crystallin of about 540 kDa. However, no strong interaction between alphaB-crystallin and 20S proteasomes was observed. Since both proteins are localized in the cytoplasm, the interaction between alphaB-crystallin and C8/alpha7 subunit might affect the assembly of the proteasome complex or facilitate the degradation of unfolded proteins bound to alphaB-crystallin.

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic alpha-helix.

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.

The lack of chaperonelike activity of Caenorhabditis elegans Hsp12.2 cannot be restored by domain swapping with human alphaB-crystallin.

The small heat shock proteins Hsp12.2 and alphaB-crystallin differ in that the former occurs as tetramers, without chaperonelike activity, whereas the latter forms multimers and is a good chaperone. To investigate whether the lack of chaperone activity of Hsp12.2 is primarily due to its tetrameric structure or rather to intrinsic sequence features, we engineered chimeric proteins by swapping the N-terminal, C-terminal, and tail regions of Hsp12.2 and alphaB-crystallin, designated as n-c-t and N-C-T, respectively. Three of the chimeric sHsps, namely N-c-T, n-c-T, and N-C-t, showed nativelike secondary and quaternary structures as measured by circular dichroism and gel permeation chromatography. Combining the conserved alpha-crystallin domain of Hsp12.2 with the N-terminal and tail regions of alphaB-crystallin (N-c-T) resulted in multimeric complexes, but did not restore chaperonelike activity. Replacing the tail region of Hsp12.2 with that of alphaB-crystallin (n-c-T) did not alter the tetrameric structure and lack of chaperone activity. Similarly, providing alphaB-crystallin with the tail of Hsp12.2 (N-C-t) did not substantially influence the multimeric complex size, but it reduced the chaperoning ability, especially for small substrates. These results suggest that the conserved alpha-crystallin domain of Hsp12.2 is intrinsically unsuitable to confer chaperonelike activity and confirms that the tail region in alphaB-crystallin modulates chaperonelike capacity in a substrate-dependent manner.

Characteristics of super alphaA-crystallin, a product of in vitro exon shuffling.

alphaA-Crystallin, a small heat shock protein with chaperone-like activity, forms dynamic multimeric complexes. Recently we described the spontaneous generation of a mutant protein (super alphaA-crystallin) by exon duplication arisen via exon shuffling confirming a classic hypothesis by Gilbert [Nature 271 (1978) 501]. Comparison of super alphaA-crystallin, which is viable in a mouse skeletal muscle cell line, with normal alphaA-crystallin shows that it has diminished thermostability, increased exposure of hydrophobic patches, a larger complex size and lost its chaperone activity. However, super alphaA-crystallin subunits exchange as readily between complexes as does normal alphaA-crystallin. These data indicate that chaperone-like activity may vanish independent of subunit hydrophobicity and exchangeability.

The molecular chaperone alphaB-crystallin enhances amyloid beta neurotoxicity.

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.

HspB3, the most deviating of the six known human small heat shock proteins.

From the alignment of 14 EST clones, the cDNA sequence of a novel human small heat shock protein (sHsp), called HspB3, could be deduced. The 3' part of the HspB3 cDNA is 99% identical to that of the previously reported HspL27 cDNA (W.Y. Lam, S.K. Wing Tsui, P.T. Law, S.C. Luk, K.P. Fung, C.Y. Lee, M.M. Waye, Isolation and characterization of a human heart cDNA encoding a new member of the small heat shock protein family-HSPL27, Biochim. Biophys. Acta 1314 (1996) 120-124). We argue that the HspB3 cDNA sequence is a corrected version of the HspL27 cDNA. The HspB3 cDNA is 742 bp long and contains an open reading frame specifying a polypeptide of 150 amino acid residues. Among the six known human sHsps it is evident that HspB3 is the most deviating one, having a unique N-terminal domain and essentially lacking a C-terminal extension. Northern blot analysis shows that in smooth muscle tissue the cDNA hybridizes with mRNA of about 0.9 kb.

Negative charges in the C-terminal domain stabilize the alphaB-crystallin complex.

alphaB-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserved sequence of 80-100 residues, which constitutes the putative C-terminal domain. Like other sHsps, alphaB-crystallin forms multimeric globular complexes, often in combination with related sHsps. Here we show that in a yeast two-hybrid system, alphaB-crystallin can specifically interact with itself as well as with alphaA-crystallin and Hsp27. Analyses of the separate domains show that the conserved C-terminal domain (CalphaB) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the CalphaB domain was used in a two-hybrid screen as bait to select randomly mutated CalphaB mutants. In this way we obtained nine mutants that were still able to interact with wild-type CalphaB despite the presence of up to 15 replacements. Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nine replacements. In binding CalphaB mutants, lysine residues were most often replaced by glutamic acid residues, and in non-binding CalphaB mutants, acidic residues were often found to be replaced by non-charged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of acidic residues. Furthermore, we observed that two homologs of alphaB-crystallin, alphaA-crystallin and Hsp27, generally interact similarly with the binding and non-binding CalphaB mutants as does alphaB-crystallin. This suggests that interactions involved in the complex formation of these three sHsps are largely comparable.

Caenorhabditis elegans small heat-shock proteins Hsp12.2 and Hsp12.3 form tetramers and have no chaperone-like activity.

Four 12.2-12.6 kDa small heat-shock proteins (sHSPs) of Caenorhabditis elegans are the smallest known members of the sHSP family. They essentially comprise the characteristic C-terminal 'alpha-crystallin domain' of the sHSPs, having a very short N-terminal region, and lacking a C-terminal tail. Recombinant Hsp12.2 and 12.3 are characterized here. Far-UV CD spectra reveal, as for other sHSPs, predominantly a beta-sheet structure. By gel permeation and crosslinking, they are the first sHSPs shown to occur as tetramers, rather than forming the usual large multimeric complexes. Exceptionally, too, both appear devoid of in vitro chaperone-like abilities. This supports the notion that tetramers are the building blocks of sHSP complexes, and that higher multimer formation, mediated through the N-terminal domains, is a prerequisite for chaperone-like activity.

The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs.

HIV-1 Rev protein directs nuclear export of pre-mRNAs and mRNAs containing its binding site, the Rev response element (RRE). To define how Rev acts, we used conjugates between bovine serum albumin (BSA) and peptides comprising the Rev activation domain (BSA-R). BSA-R inhibited Rev-mediated nuclear RNA export, whereas a mutant activation domain peptide conjugate did not. BSA-R did not affect the export of mRNA, tRNA, or ribosomal subunits, but did inhibit export of 5S rRNA and spliceosomal U snRNAs. BSA-R was itself exported from the nucleus in an active, saturable manner. Thus, the Rev activation domain constitutes a nuclear export signal that redirects RRE-containing viral RNAs to a non-mRNA export pathway.

Nuclear retention of RNA as a mechanism for localization.

Two mutant RNAs, one derived from tRNA(imet), the second from U1 snRNA, that are defective in export from the nucleus to the cytoplasm have been studied. In both cases, the RNAs are shown to be transport competent but prevented from leaving the nucleus by interaction with saturable binding sites. This contradicts previous hypotheses to explain the behavior of the tRNA mutant, and highlights a general problem in using mutant RNAs to study nuclear export. In the case of these mutants, it is argued that nuclear retention is likely to be artifactual. However, the additional example of U6 snRNA is described. In this case, nuclear retention appears to be a physiological mechanism by which intranuclear localization is achieved. Evidence that the site of interaction with the La protein in U6 snRNA is important for its nuclear retention is presented.

Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif.

An essential yeast protein, Nup145p, is identified via its genetic interaction with the nucleoporin Nsp1p. Nup145p contains GLFG repeats and localizes to nuclear pores. Depletion of Nup145p in vivo leads rapidly to nuclear retention of polyadenylated RNAs and more slowly to cytoplasmic accumulation of a nuclear reporter protein. A stretch of 140 amino acids within Nup145p is conserved in two other yeast nucleoporins, Nup116p and Nup100p, and in an uncharacterized C. elegans protein. Genetic experiments in yeast reveal that the three copies of the motif carry out an essential, redundant function. Fragments of Nup145p and Nup116p including this motif bind specifically to homopolymeric RNAs in vitro. Nup145p, Nup116p, and Nup100p thus represent a novel class of nucleoporins involved in nucleocytoplasmic transport.

Nuclear export of different classes of RNA is mediated by specific factors.

Various classes of RNA are exported from the nucleus to the cytoplasm, including transcripts of RNA polymerase I (large ribosomal RNAs), II (U-rich small nuclear RNAs [U snRNAs], mRNAs), and III (tRNAs, 5S RNA). Here, evidence is presented that some steps in the export of various classes of nuclear RNA are mediated by specific rather than common factors. Using microinjection into Xenopus oocytes, it is shown that a tRNA, a U snRNA, and an mRNA competitively inhibit their own export at concentrations at which they have no effect on the export of heterologous RNAs. While the export of both U snRNAs and mRNAs is enhanced by their 7-methyl guanosine cap structures, factors recognizing this structure are found to be limiting in concentration only in the case of U snRNAs. In addition to the specific factors, evidence for steps in the export process that may be common to at least some classes of RNA are provided by experiments in which synthetic homopolymeric RNAs are used as inhibitors.

The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase.

The human U1 snRNP-specific U1A protein autoregulates its production by binding its own pre-mRNA and inhibiting polyadenylation. The mechanism of this regulation has been elucidated by in vitro studies. U1A protein is shown not to prevent either binding of cleavage and polyadenylation specificity factor (CPSF) to its recognition sequence (AUUAAA) or to prevent cleavage of U1A pre-mRNA. Instead, U1A protein bound to U1A pre-mRNA inhibits both specific and nonspecific polyadenylation by mammalian, but not by yeast, poly(A) polymerase (PAP). Domains are identified in both proteins whose removal uncouples the polyadenylation activity of mammalian PAP from its inhibition via RNA-bound U1A protein. Finally, U1A protein is shown to specifically interact with mammalian PAP in vitro. The possibility that this interaction may reflect a broader role of the U1A protein in polyadenylation is discussed.