Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.     

Carbohydrate-Binding Proteins in the Leech: I. Isolation and Characterization of Lactose-Binding Proteins

Abstract: Three lactose-binding proteins with apparent molecular masses of 16, 35, and 63 kDa [leech lectin 16, 35, and 63 (LL16, LL35, and LL63, respectively)] were isolated from leech membranes. Polyclonal antibodies raised against LL35 cross-reacted with LL16 and LL63, indicating that all three lectins were immunologically related. These leech lectins, however, can be subdivided into two groups based on their tissue distributions and binding affinities for galactose derivatives. LL16 and LL35 are endogenous to the leech's CNS, whereas LL63 is only present in peripheral organs. LL16 and LL35, found in the CNS, bind both the α and β anomers of methylgalactose, whereas the peripheral lectin LL63 binds only the β form. LL35 and LL63 also differ in their binding affinities for galactosamine and N -acetylgalactosamine. The binding activity of LL35 was calcium independent and active over a wide pH range. Triton X-100 and 2-mercaptoethanol were necessary to recover LL35 binding activity during extraction. These characteristics strongly suggest that LL35 is another member of the calcium-independent galactose/lactose-specific lectins previously described in vertebrates and recently demonstrated in sponges and nematodes. Because a single leech expresses up to 100 µg of LL35, this leech lectin is readily amenable to structural and functional analysis.     

Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.     

Tracing Primordial Protein Evolution through Structurally Guided Stepwise Segment Elongation

The understanding of how primordial proteins emerged has been a fundamental and longstanding issue in biology and biochemistry. For a better understanding of primordial protein evolution, we synthesized an artificial protein on the basis of an evolutionary hypothesis, segment-based elongation starting from an autonomously foldable short peptide. A 10-residue protein, chignolin, the smallest foldable polypeptide ever reported, was used as a structural support to facilitate higher structural organization and gain-of-function in the development of an artificial protein. Repetitive cycles of segment elongation and subsequent phage display selection successfully produced a 25-residue protein, termed AF.2A1, with nanomolar affinity against the Fc region of immunoglobulin G. AF.2A1 shows exquisite molecular recognition ability such that it can distinguish conformational differences of the same molecule. The structure determined by NMR measurements demonstrated that AF.2A1 forms a globular protein-like conformation with the chignolin-derived β-hairpin and a tryptophan-mediated hydrophobic core. Using sequence analysis and a mutation study, we discovered that the structural organization and gain-of-function emerged from the vicinity of the chignolin segment, revealing that the structural support served as the core in both structural and functional development. Here, we propose an evolutionary model for primordial proteins in which a foldable segment serves as the evolving core to facilitate structural and functional evolution. This study provides insights into primordial protein evolution and also presents a novel methodology for designing small sized proteins useful for industrial and pharmaceutical applications.     

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from ¹⁵N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are α-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. ¹⁵N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.     

Identification of functional subclasses in the DJ-1 superfamily proteins

Genomics has posed the challenge of determination of protein function from sequence and/or 3-D structure. Functional assignment from sequence relationships can be misleading, and structural similarity does not necessarily imply functional similarity. Proteins in the DJ-1 family, many of which are of unknown function, are examples of proteins with both sequence and fold similarity that span multiple functional classes. THEMATICS (theoretical microscopic titration curves), an electrostatics-based computational approach to functional site prediction, is used to sort proteins in the DJ-1 family into different functional classes. Active site residues are predicted for the eight distinct DJ-1 proteins with available 3-D structures. Placement of the predicted residues onto a structural alignment for six of these proteins reveals three distinct types of active sites. Each type overlaps only partially with the others, with only one residue in common across all six sets of predicted residues. Human DJ-1 and YajL from Escherichia coli have very similar predicted active sites and belong to the same probable functional group. Protease I, a known cysteine protease from Pyrococcus horikoshii, and PfpI/YhbO from E. coli, a hypothetical protein of unknown function, belong to a separate class. THEMATICS predicts a set of residues that is typical of a cysteine protease for Protease I; the prediction for PfpI/YhbO bears some similarity. YDR533Cp from Saccharomyces cerevisiae, of unknown function, and the known chaperone Hsp31 from E. coli constitute a third group with nearly identical predicted active sites. While the first four proteins have predicted active sites at dimer interfaces, YDR533Cp and Hsp31 both have predicted sites contained within each subunit. Although YDR533Cp and Hsp31 form different dimers with different orientations between the subunits, the predicted active sites are superimposable within the monomer structures. Thus, the three predicted functional classes form four different types of quaternary structures. The computational prediction of the functional sites for protein structures of unknown function provides valuable clues for functional classification.     

Primary sequence that determines the functional overlap between mitochondrial heat shock protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can "make or break" mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.     

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.     

The human TREX2 3' -> 5'-exonuclease structure suggests a mechanism for efficient nonprocessive DNA catalysis

The 3' --> 5'-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3'-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3' termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by approximately 100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3'-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by approximately 10(5)-fold, confirming their catalytic role.     

Hirudo medicinalis: A Platform for Investigating Genes in Neural Repair

We have used the nervous system of themedicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlikemammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity.We have constructed subtractive cDNAprobes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell.This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP tsynthase are upregulated at 24 h postinjury in leech nerve cord.To investigate the functional role of regulated genes in neuron regrowthwe are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1.Our approach thus identifies genes regulated at different times after injury thatmay underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.     

Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain under Physiological Conditions

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.     

Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5 M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation–rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a “molecular shield”. Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.     

The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2

The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Several proteins showing sequence similarity to Rad51 have previously been identified in both yeast and human cells. In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange. Here, we report the purification of one of the representatives of the RAD51 family in human cells. We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence. We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2. By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct. Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.     

Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.     

Identification of molecules in leech extracellular matrix that promote neurite outgrowth

The molecular composition of the substrate is of critical importance for neurite extension by isolated identified leech nerve cells in culture. One substrate upon which rapid growth occurs in defined medium is a cell-free extract of extracellular matrix (ECM) that surrounds the leech central nervous system (CNS). Here we report the co-purification of neurite-promoting activity with a laminin-like molecule. High molecular mass proteins from leech ECM purified by gel filtration exhibited increased specific activity for promoting neurite outgrowth. The most active fractions contained three major polypeptide bands of ca. 340, 250 and 220 kDa. Electron microscopy of rotary-shadowed samples showed three macromolecules, one of which had a cross-shaped structure similar to vertebrate laminin. A second six-armed molecule resembled vertebrate tenascin and a third rod-like molecule resembled vertebrate collagen type IV. The most active fractions contained a protein of ca. 1 MDa on non-reducing gels with disulphide-linked subunits of ca. 220 and 340 kDa, with cross-shaped laminin-like molecules. We conclude that a laminin-like molecule represents a major neurite promoting component present in leech ECM. The experiments represent a first step in determining the location of leech laminin within the CNS and assessing its role in neurite outgrowth during development and regeneration.     

Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ)

Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.     

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

Autotransporter proteins: novel targets at the bacterial cell surface

Autotransporter proteins constitute a family of outer membrane/secreted proteins that possess unique structural properties that facilitate their independent transport across the bacterial membrane system and final routing to the cell surface. Autotransporter proteins have been identified in a wide range of Gram-negative bacteria and are often associated with virulence functions such as adhesion, aggregation, invasion, biofilm formation and toxicity. The importance of autotransporter proteins is exemplified by the fact that they constitute an essential component of some human vaccines. Autotransporter proteins contain three structural motifs: a signal sequence, a passenger domain and a translocator domain. Here, the structural properties of the passenger and translocator domains of three type Va autotransporter proteins are compared and contrasted, namely pertactin from Bordetella pertussis, the adhesion and penetration protein (Hap) from Haemophilus influenzae and Antigen 43 (Ag43) from Escherichia coli. The Ag43 protein is described in detail to examine how its structure relates to functional properties such as cell adhesion, aggregation and biofilm formation. The widespread occurrence of autotransporter-encoding genes, their apparent uniform role in virulence and their ability to interact with host cells suggest that they may represent rational targets for the design of novel vaccines directed against Gram-negative pathogens.