N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.     

Function and structure of N-terminal and C-terminal domains of calcineurin B subunit

Calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca2+-binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca2+ binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively; DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca2+ to CNB, DC or DN leads to exposure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity.     

Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces cerevisiae

The molecular chaperone Hsp90 is a regulatory component of some key signalling proteins in the cytosol of eukaryotic cells. For some of these functions, its interaction with co-chaperones is required. Limited proteolysis defined stable folded units of Hsp90. Both an N-terminal (N210) and a C-terminal (262C) fragment interact with non-native substrate proteins in vitro, but with different specificity and ATP dependence. Here, we analysed the functional properties of these Hsp90 fragments in vivo and in vitro. We determined their influence on the general viability and cell growth of Saccharomyces cerevisiae. Expression of N210 or 262C resulted in a dominant-negative phenotype in several yeast strains tested. Their expression was not toxic, but inhibited cell growth. Further, both were unable to restore viability to Hsp90-depleted cells. In addition, N210 and 262C influence the maturation of Hsp90 substrates, such as the glucocorticoid receptor and pp60v-Src kinase. Specifically, 262C forms partially active chaperone complexes, leading to an arrest of the chaperoned substrate at a certain stage of its maturation cycle. This demonstrates the requirement of a sophisticated and cofactor-regulated interplay between N- and C-terminal activities for Hsp90 function in vivo.     

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Mesothelial cells activate the plasma kallikrein-kinin system during pleural inflammation

Abstract Pleural inflammation underlies the formation of most exudative pleural effusions and the plasma kallikrein-kinin system (KKS) is known to contribute. Mesothelial cells are the predominant cell type in the pleural cavity, but their potential role in plasma KKS activation and BK production has not been studied. Bradykinin concentrations were higher in pleural fluids than the corresponding serum samples in patients with a variety of diseases. Bradykinin concentrations did not correlate with disease diagnosis, but were elevated in exudative effusions. It was demonstrated, using a range of primary and transformed mesothelial and mesothelioma cell lines, that cells assembled high molecular weight kininogen and plasma prekallikrein to liberate bradykinin, a process inhibited by novobiocin, a heat shock protein 90 (HSP90) inhibitor, cysteine, bradykinin and protamine sulphate. Of the common plasma prekallikrein activators, mesothelial cells expressed HSP90, but not prolylcarboxypeptidase or Factor XII. Calcium mobilisation was induced in some mesothelium-derived cell lines by bradykinin. Des-Arg(9)-bradykinin was inactive, indicating that mesothelial cells are responsive to bradykinin, mediated via the bradykinin receptor subtype 2. In summary, pleural mesothelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and may contribute to KKS-mediated inflammation in pleural disease.     

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.     

Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1-48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-mediated adherence, arguing that the C-terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram-negative bacterial exoproteins.     

Maltose chemotaxis involves residues in the N-terminal and C-terminal domains on the same face of maltose-binding protein.

The periplasmic maltose-binding protein (MBP) of Escherichia coli is the recognition component of the maltose chemoreceptor and of the active transport system for maltose. It interacts with the Tar chemotactic signal transducer and the integral cytoplasmic-membrane components (the MalF and MalG proteins) of the maltose transport system. Maltose binds in a cleft between the globular N-terminal and C-terminal domains of MBP, which are connected by a moveable hinge. The two domains undergo a large motion relative to one another as the protein moves from the open, unbound state to the closed, ligand-bound state. We generated, by doped-primer mutagenesis, amino acid substitutions that specifically disrupt the chemotactic function of MBP. These substitutions cluster in two well-defined regions that are nearly contiguous on the surface of MBP in its closed conformation. One region is in the N-terminal domain and one is in the C-terminal domain. The distance between the two regions is expected to change substantially as the protein goes from the open to the closed form. These results support a model in which ligand binding brings two recognition sites on MBP into the proper spatial relationship to interact with complementary sites on Tar. Mutations in MBP that appear to cause defects in interaction with MalF and MalG are distributed differently from mutations that primarily affect maltose taxis. We conclude that the regions of MBP that contact Tar and those that contact MalF and MalG are adjacent on the face of the protein opposite the hinge connecting the two domains and that those regions are largely, although perhaps not entirely, distinct.     

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.     

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.     

DNA binding of PriA protein requires cooperation of the N-terminal D-loop/arrested-fork binding and C-terminal helicase domains

PriA protein is essential for RecA-dependent DNA replication induced by stalled replication forks in Escherichia coli. PriA is a DEXH-type DNA helicase, ATPase activity of which depends on its binding to structured DNA including a D-loop-like structure. Here, we show that the N-terminal 181-amino acid polypeptide can form a complex with D-loop in gel shift assays and have identified a unique motif present in the N-terminal segment of PriA that plays a role in its DNA binding. We have also identified residues in the C terminus proximal helicase domain essential for D-loop binding. PriA proteins mutated in this domain do not bind to D-loop, despite the presence of the N-terminal DNA-binding motif. Those mutants that cannot bind to D-loop in vitro do not support a recombination-dependent mode of DNA replication in vivo, indicating that binding to a D-loop-like structure is essential for the ability of PriA to initiate DNA replication and repair from stalled replication forks. We propose that binding of the PriA protein to stalled replication forks requires proper configuration of the N-terminal fork-recognition and C-terminal helicase domains and that the latter may stabilize binding and increase binding specificity.     

Characterization of C- and N-terminal domains of Aquifex aeolicus MutL endonuclease: N-terminal domain stimulates the endonuclease activity of C-terminal domain in a zinc-dependent manner

DNA MMR (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3'-5' proofreading activity associated with replicative DNA polymerases. MutL proteins homologous to human PMS2 are the endonucleases that introduce the entry point of the excision reaction. Deficiency in PMS2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. Although recent studies revealed that the CTD (C-terminal domain) of MutL harbours weak endonuclease activity, the regulatory mechanism of this activity remains unknown. In this paper, we characterize in detail the CTD and NTD (N-terminal domain) of aqMutL (Aquifex aeolicus MutL). On the one hand, CTD existed as a dimer in solution and showed weak DNA-binding and Mn2+-dependent endonuclease activities. On the other hand, NTD was monomeric and exhibited a relatively strong DNA-binding activity. It was also clarified that NTD promotes the endonuclease activity of CTD. NTD-mediated activation of CTD was abolished by depletion of the zinc-ion from the reaction mixture or by the substitution of the zinc-binding cysteine residue in CTD with an alanine. On the basis of these results, we propose a model for the intramolecular regulatory mechanism of MutL endonuclease activity.     

The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains

Background

Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear.

Results

Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues ⁷⁵GCRHSRIGVTRQRRAR⁹⁰, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr^75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA.

Conclusions

For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.