The structure of human interferon-β: implications for activity

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-β (HuIFN-β), in particular in relation to its activity. The recently determined crystal structure of HuIFN-β provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-β, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-β. Received 12 June 1998; received after revision 16 July 1998; accepted 16 July 1998     

Common structural traits for cystine knot domain of the TGFβ superfamily of proteins and three-fingered ectodomain of their cellular receptors

The transforming growth factor-β (TGFβ) superfamily of proteins and their receptors are crucial developmental factors for all metazoan organisms. Cystine-knot (CK) motif is a spatial feature of the TGFβ superfamily of proteins whereas the extra-cellular domains (ectodomains) of their respective receptors form three-fingered protein domain (TFPD), both stabilized by tight cystine networks. Analyses of multiple sequence alignments of these two domains encoded in various genomes revealed that the cystines forming the CK and TFPD folds are conserved, whereas the remaining polypeptide patches are diversified. Orthologues of the human TGFβs and their respective receptors expressed in diverse vertebrates retain high sequence conservation. Examination of 3D structures of various TGFβ factors bound to their receptors have revealed that the CK and TFPD domains display several similar spatial traits suggesting that these two different protein folds might have been acquired from a common ancestor.     

"Add-on" domains of<Emphasis Type="Italic"> Drosophila</Emphasis> β1,4-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminyltransferase B in the stem region and its pilot protein

The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3–4 and 5–6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.     

Production of functional rat liver PSP protein in Escherichia coli

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH₂-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998     

Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function

Tissue microfibrils contain fibrillin-1 as a major constituent. Microfibrils regulate bioavailability of TGFβ superfamily growth factors and are structurally crucial in the ocular zonule. FBN1 mutations typically cause the Marfan syndrome, an autosomal dominant disorder manifesting with skeletal overgrowth, aortic aneurysm, and lens dislocation (ectopia lentis). Infrequently, FBN1 mutations cause dominantly inherited Weill–Marchesani syndrome (WMS), isolated ectopia lentis (IEL), or the fibrotic condition, geleophysic dysplasia (GD). Intriguingly, mutations in ADAMTS [a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] family members phenocopy these disorders, leading to recessive WMS (ADAMTS10), WMS-like syndrome (ADAMTS17), IEL (ADAMTSL4 and ADAMTS17) and GD (ADAMTSL2). An ADAMTSL2 founder mutation causes Musladin–Lueke syndrome, a fibrotic disorder in beagle dogs. The overlapping disease spectra resulting from fibrillin-1 and ADAMTS mutations, interaction of ADAMTS10 and ADAMTSL2 with fibrillin-1, and evidence that these ADAMTS proteins accelerate microfibril biogenesis, constitutes a consilience suggesting that some ADAMTS proteins evolved to provide a novel mechanism regulating microfibril formation and consequently cell behavior.     

G proteins as drug targets

The structure and function of heterotrimeric G protein subunits is known in considerable detail. Upon stimulation of a heptahelical receptor by the appropriate agonists, the cognate G proteins undergo a cycle of activation and deactivation; the α-subunits and the βγ-dimers interact sequentially with several reaction partners (receptor, guanine nucleotides and effectors as well as regulatory proteins) by exposing appropriate binding sites. For most of these domains, low molecular weight ligands have been identified that either activate or inhibit signal transduction. These ligands include short peptides derived from receptors, G protein subunits and effectors, mastoparan and related insect venoms, modified guanine nucleotides, suramin analogues and amphiphilic cations. Because compounds that act on G proteins may be endowed with new forms of selectivity, we propose that G protein subunits may therefore be considered as potential drug targets. Received 18 September 1998; received after revision 6 November 1998; accepted 11 November 1998     

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000     

Substrate binding and catalytic mechanism of class B β-lactamases: a molecular modelling study

Increased resistance to β-lactam antibiotics is mainly due to β-lactamases whose production by pathogenic bacteria makes their broad activity spectrum especially frightening. X-ray structures of several zinc β-lactamases have revealed the coordination of the two metal ions, but their mode of action remains unclear. Geometry optimisation of stable complexes along the reaction pathway of benzylpenicillin hydrolysis highlighted a proton shuttle occurring from D120 of the Bacillus cereus β-lactamase to the β-lactam nitrogen via Zn2 which is central to the network. First, the Zn1 ion has a structural role maintaining Zn-bound waters, WAT1 and WAT2, either directly or through the Zn1 tetrahedrally coordinated histidine ligands. The Zn2 ion has a more catalytic role, stabilising the tetrahedral intermediate, accepting the β-lactam nitrogen atom as a ligand. The role of Zn2 and the flexibility in the coordination geometry of both Zn ions is of crucial importance for catalysis. Received 14 August 2001; received after revision 19 October 2001; accepted 30 October 2001     

SET domain protein lysine methyltransferases: Structure, specificity and catalysis

Site- and state-specific lysine methylation of histones is catalyzed by a family of proteins that contain the evolutionarily conserved SET domain and plays a fundamental role in epigenetic regulation of gene activation and silencing in all eukaryotes. The recently determined three-dimensional structures of the SET domains from chromosomal proteins reveal that the core SET domain structure contains a two-domain architecture, consisting of a conserved anti-parallel β-barrel and a structurally variable insert that surround a unusual knot-like structure that comprises the enzyme active site. These structures of the SET domains, either in the free state or when bound to cofactor S-adenosyl-L-homocysteine and/or histone peptide, mimicking an enzyme/cofactor/substrate complex, further yield the structural insights into the molecular basis of the substrate specificity, methylation multiplicity and the catalytic mechanism of histone lysine methylation. Received 10 June 2006; accepted 22 August 2006     

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Resistant penicillin-binding proteins

Low-affinity penicillin-binding proteins (PBPs), which participate in the β-lactam resistance of several pathogenic bacteria, have different origins. Natural transformation and recombination events with DNA acquired from neighbouring intrinsically resistant organisms are responsible for the appearance of mosaic genes encoding two or three low-affinity PBPs in highly resistant strains of transformable microorganisms such as Neisseria and Streptococcus pneumoniae. Methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal strains possess the mecA determinant gene, which probably evolved within the Staphylococcus genus from a closely related and physiologically functional gene that was modified by point mutations. The expression of mecA is either inducible or constitutive. A stable high-level resistant phenotype requires the synthesis of a normally constituted peptidoglycan. Enterococci have a natural low susceptibility to β-lactams related to the presence of an intrinsic low-affinity PBP. Highly resistant enterococcal strains overexpress this PBP and/or reduce its affinity.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Cytolytic and K+ channel blocking activities of β-KTx and scorpine-like peptides purified from scorpion venoms

Among the scorpion venom components whose function are poorly known or even show contrasting pharmacological results are those called “orphan peptides”. The most widely distributed are named β-KTx or scorpine-like peptides. They contain three disulfide bridges with two recognizable domains: a freely moving N-terminal amino acid sequence and a tightly folded C-terminal region with a cysteine-stabilized α/β (CS-αβ) motif. Four such peptides and three cloned genes are reported here. They were assayed for their cytolytic, antimicrobial and K ⁺ channel-blocking activities. Two main characteristics were found: the existence of an unusual structural and functional diversity, whereby the full-length peptide can lyse cells or kill microorganisms, and a C-terminal domain containing the CS-αβ motif that can block K ⁺ channels. Furthermore, sequence analyses and phylogenetic reconstructions are used to discuss the evolution of this type of peptide and to highlight the versatility of the CS-αβ structures. Received 13 August 2007; received after revision 30 October 2007; accepted 2 November 2007     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

Chimeras of human lysozyme and α-lactalbumin: an interesting tool for studying partially folded states during protein folding

Protein folding is an extremely active field of research where biology, chemistry, computer science and physics meet. Although the study of protein-folding intermediates in general and equilibrium intermediates in particular has grown considerably in recent years, many questions regarding the conformational state and the structural features of the various partially folded intermediate states remain unanswered. Performing kinetic measurements on proteins that have had their structures modified by site-directed mutagenesis, the so-called protein-engineering method, is an obvious way to gain fine structural information. In the present review, this method has been applied to a variety of proteins belonging to the lysozyme/α-lactalbumin family. Besides recombinants obtained by point mutations of individual critical residues, chimeric proteins in which whole structural elements (10 – 25 residues) from α-lactalbumin were inserted into a human lysozyme matrix are examined. The conformational properties of the equilibrium intermediate states are discussed together with the structural characterization of the partially unfolded states encountered in the kinetic folding pathway. Received 28 May 1998; received after revision 6 July 1998; accepted 6 July 1998     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

The mitochondrial processing peptidase: Function and specificity

Targeting signals of mitochondrial precursors are cleaved in the matrix during or after import by the mitochondrial processing peptidase (MPP). This enzyme consists of two nonidentical- and-subunits each of molecular weight of about 50 kDa. In mammals and fungi, MPP is soluble in the matrix, whereas in plants the enzyme is part of the cytochromebc ₁ complex. MPP is a metalloendopeptidase which has been classified as a member of the pitrilysin family on the basis of the HXXEHX₇₆E zinc-binding motif present in-MPP. Both subunits of MPP are required for processing activity. The-subunit of MPP, which probably recognizes a three-dimensional motif adopted by the presequence, presents the presequence to-MPP, which carries the catalytic active site. MPP acts as an endoprotease on chemically synthesized peptides corresponding to mitochondrial presequences. Matrix-targeting signals and MPP cleavage signals seem to be distinct, although the two signals may overlap within a given presequence. The structural element helix-turn-helix, that cleavable presequences adopt in a membrane mimetic environment, may be required for processing but is not sufficient for proteolysis. Binding of the presequence by-MPP tolerates a high degree of mutations of the presequence.-MPP may present a degenerated cleavage site motif to-MPP in an accessible conformation for processing. The conformation of mitochondrial presequences bound to MPP remains largely unknown.     

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys₆-Cys₂₁, Cys₁₃-Cys₃₀, Cys₂₀-Cys₄₈, Cys₃₂-Cys₄₆). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. Received 23 July 2001; accepted 31 July 2001     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

Natriuretic peptide hormones promote radial water movements from the xylem of Tradescantia shoots

Immunological evidence suggests that plants, like vertebrates, contain natriuretic peptides (NPs) and that rat atrial NP (rANP) binds specifically to plant membranes and promotes concentration and conformation-dependent stomatal opening. Stomatal opening and specific increases in cGMP levels were also observed in response to immunoreactive plant NP (irPNP). Here we report that both 1 μM rANP and irPNP (100 ng total protein/100 μL) significantly increase radial water movements out of the xylem of shoots of Tradescantia multiflora. Enhanced radial water movements are also observed in response to the cell permeant cGMP analogue 8-Br-cGMP (100 nM). The water channel inhibitor mercuric chloride (HgCl₂) significantly inhibits radial water movements at concentrations of 50 μM, while the presence of 10 μM 2-hydroxyethylmercaptoethanol (ME) prevents the inhibitory effect of the mercurial. The guanylate cyclase inhibitor LY 83583 at a concentration of 20 μM and sodium azide (NaN₃) at concentrations of ≥ 1 μM both also reduce radial water movements. We therefore conclude that the regulation of radial water movement out of the xylem involves modulation of cGMP levels, water channels and respiration-dependent processes. In addition, we propose that NPs have a critical role to play in radial water movements out of the xylem and speculate that as in vertebrates, NP effects might, at least in part, be mediated via the regulation of guanylate cyclases and water channels. Received 15 June 1998; received after revision 7 August 1998; accepted 26 August 1998