Volume 387, Issue 3

This review summarizes the knowledge on striated domains, which are ordered assemblies of transmembrane peptides and lipids under gel-state conditions. The structure, mechanism of function and utility of this system as a model for domain formation is described, resulting in a molecular description of the domains and a discussion on the relevance of these insights for the function/formation and structure of similar domains in biological membranes.

The human kallikrein locus on chromosome 19 consists of 15 genes encoding serine proteases. Here we review studies on their evolution, which demonstrate that there are several taxon-unique KLK1 paralogs in mouse, rat and horse, but not in primates and many other mammals. Furthermore, the duplication yielding KLK2 and prostate-specific antigen (KLK3) appears to be specific to primates, but a functional progenitor to KLK2 is expressed in the dog prostate. The linkage to a locus of possible regulatory protease inhibitors on chromosome 20 is discussed, as is the potential role of the kallikrein locus in innate immunity.

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo . This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.

Mutation detection and mismatch repair investigations based on heteroduplex formation require a linear DNA structure. DNA branching, described previously under physiological conditions, has been analysed in the heteroduplex formation process. Symmetrical χ-structures were detected after heteroduplex formation by gel electrophoresis and electron microscopy. Buffer composition, DNA concentration and duplex end-sequences influence DNA branching. Duplexes with homologous central regions but non-complementary ends do not form hybrid heteroduplexes or hybrid Holliday junctions. Our results explain the requirements for efficient heteroduplex formation, which were previously determined empirically: special solution composition, optimal DNA concentration and GC clamps. This provides the theoretical background for further optimisation of the procedure.

In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.

The human gene BDP1 , localized on chromosome 5q13 in close proximity to the spinal muscular atrophy determining gene SMN , encodes a large protein consisting of 2254 amino acids (aa). In the first third of the gene, the subunit of the RNA polymerase III (Pol III) transcription factor complex (TFIIIBα/β) is encoded. To further characterize the function of BDP1, we carried out a yeast two-hybrid screen using various parts of BDP1. With the clone BDP1-(1–640) we identified a novel interaction partner, ZNF297B. The ZNF297B gene is localized on chromosome 9q24 and encodes a zinc finger protein of 467 aa possessing the typical structure of a transcription factor. The interaction found in yeast was confirmed by co-immunoprecipitation and refined to the N-terminal region of ZNF297B-(1–127) containing the BTB/POZ domain and the N-terminal end of BDP1-(1–299). The ZNF297B transcript is 5.7 kb in length and ubiquitously expressed, with highest levels found in muscles. Immunofluorescence staining revealed a speckled pattern in the nuclei of HEK293 cells. Due to the essential role of BDP1 in Pol III transcription, we propose that ZNF297B may also regulate these transcriptional pathways.

In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have non-additive positive stability interaction energies that are consistent with exquisite tight core packing and rearrangements of van der Waals' interactions in the protein interior, even after drastic deleterious substitutions.

A key molecular event in prion diseases is the conversion of cellular prion protein (PrP c ) into an abnormal misfolded conformer (PrP sc ). The PrP c N-terminal domain plays a central role in PrP c functions and in prion propagation. Because mammalian PrP c is found as a full-length and N-terminally truncated form, we examined the presence and amount of PrP c C-terminal fragment in the brain of different species. We found important variations between primates and rodents. In addition, our data show that the PrP c fragment is present in detergent-resistant raft domains, a membrane domain of critical importance for PrP c functions and its conversion into PrP sc .

Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2- O -α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-α( R )-3-hydroxytetradecanoyl-( R )-3-hydroxytetradecanoate (RL-2,2 14 ) produced by Burkholderia ( Pseudomonas ) plantarii . RL-2,2 14 is structurally very similar to a rhamnolipid exotoxin from Pseudomonas aeruginosa and identical to the rhamnolipid of Burkholderia pseudomallei , the causative agent of melioidosis. Interestingly, RL-2,2 14 exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor α, the overproduction of which is known to cause sepsis and the septic shock syndrome. Such a property has not been noted so far for rhamnolipid exotoxins, only for bacterial endotoxins (lipopolysaccharide, LPS). Consequently, we analyzed RL-2,2 14 with respect to its pathophysiological activities as a heat-stable extracellular toxin. Like LPS, the cell-stimulating activity of the rhamnolipid could be inhibited by incubation with polymyxin B. However, immune cell activation by RL-2,2 14 does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,2 14 exhibits a variety of endotoxin-related physicochemical characteristics, such as a cubic-inverted supramolecular structure. These data are in good agreement with our conformational concept of endotoxicity: intercalation of naturally originating virulence factors into the immune cell membrane leads to strong mechanical stress on integral proteins, eventually causing cell activation.

The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.

Adenosine deaminase (ADA) plays an important role in the immune system, and its activity is composed of two kinetically distinct isozymes, ADA1 and ADA2. ADA2 is a major component of human plasma total ADA activity and ADA2 activity is significantly elevated in patients with various diseases such as HIV infection and chronic hepatitis. However, relatively little is known about ADA2 because of difficulties in purifying this enzyme. In this study we succeeded in purifying human plasma ADA2 and demonstrate the extracellular secretion of ADA2 from human peripheral blood monocytes stimulated with phorbol 12-myristate 13-acetate and calcium ionophore.

We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287–1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off -expressed mRNAs were also inhibited upon exposure to H 2 O 2 . However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H 2 O 2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas γ-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.

Calpains are a large family of Ca 2+ -dependent cysteine proteases that are ubiquitously distributed across most cell types and vertebrate species. Calpains play a role in cell differentiation, apoptosis, cytoskeletal remodeling, signal transduction and the cell cycle. The cell cycle proteins cyclin D1 and p21 KIP1 , for example, have been shown to be affected by calpains. However, the rules that govern calpain cleavage specificity are poorly understood. We report here studies on the pattern of μ-calpain proteolysis of the p19 INK4d protein, a cyclin-dependent kinase 4/6 inhibitor that negatively regulates the mammalian cell cycle. Our data show new characteristics of calpain action: μ-calpain cleaves p19 INK4d immediately after the first and second ankyrin repeats that are structurally less stable compared to the other repeats. This is in contrast to features observed so far in the specificity of calpains for their substrates. These results imply that calpain may be involved in the cell cycle by regulating the cell cycle regulatory protein turnover through CDK inhibitors and cyclins.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot™ system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the β-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot™ method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.