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The T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) is a validated immune checkpoint protein expressed on memory CD4 + T-cellls, Tregs, CD8 + T-cell and natural killer (NK) cells. ASP8374 is a fully human monoclonal immunoglobulin (Ig) G4 antibody designed to block the interaction of TIGIT with its ligands and inhibit TIGIT signaling. ASP8374 exhibited high affinity binding to TIGIT and increased interferon (IFN)-γ production of cultured peripheral blood mononuclear cells (PBMCs) in a titratable manner. When used in combination with pembrolizumab, an anti-programmed death-1 (PD-1) antibody, ASP8374 induced higher T-cell activation in vitro than either treatment alone. An anti-mouse TIGIT antibody surrogate, mSEC1, displayed anti-tumor efficacy in an MC38 syngeneic mouse tumor model alone and in combination with an anti-programmed death-ligand 1 (PD-L1) antibody. In an additional syngeneic mouse tumor model (CT26), while mSEC1 alone did not demonstrate anti-tumor efficacy, mSEC1 combined with an anti-PD-1 antibody enhanced anti-tumor efficacy above that of the anti-PD-1 antibody alone. These data provide evidence that ASP8374 has therapeutic potential for advanced malignancies., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Alzheimer's disease (AD) is a fatal progressive neurodegenerative disorder characterized by increasing loss in memory, cognition, and function of daily living. Among the many pathologic events observed in the progression of AD, changes in amyloid β peptide (Aβ) metabolism proceed fastest, and precede clinical symptoms. BACE1 (β-secretase 1) catalyzes the initial cleavage of the amyloid precursor protein to generate Aβ. Therefore inhibition of BACE1 activity could block one of the earliest pathologic events in AD. However, therapeutic BACE1 inhibition to block Aβ production may need to be balanced with possible effects that might result from diminished physiologic functions BACE1, in particular processing of substrates involved in neuronal function of the brain and periphery. Potentials for beneficial or consequential effects resulting from pharmacologic inhibition of BACE1 are reviewed in context of ongoing clinical trials testing the effect of BACE1 candidate inhibitor drugs in AD populations., Competing Interests: The author declares no conflict of interest.

We describe structure-based design, synthesis, and biological evaluation of a series of novel inhibitors bearing a pyrazole (compounds 3a-h) or a thiazole moiety (compounds 4a-e) as the P3 ligand. We have also explored Boc-β-amino-l-alanine as a novel P2 ligand. A number of inhibitors have displayed β-secretase inhibitory potency. Inhibitor 4c has shown potent BACE1 inhibitory activity, Ki=0.25nM, cellular EC50 of 194nM, and displayed good selectivity over BACE2. A model of 4c was created based upon the X-ray structure of 2-bound β-secretase which revealed critical interactions in the active site., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Alzheimer disease is intimately linked to an excess amount of amyloid-β (Aβ) in the brain. Thus, therapeutic inhibition of Aβ production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of β-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble Aβ in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long-term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid-β plaque load. We also found no accumulation of amyloid-β precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Aβ accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Aβ reduction therapy as a treatment of AD.

Structure-based design, synthesis, and biological evaluation of a series of peptidomimetic beta-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K(i) 1.8 nM) and cellular (IC(50)=1 nM in Chinese hamster ovary cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma A beta) in transgenic mice. The X-ray structure of protein-ligand complex of memapsin 2 revealed critical interactions in the memapsin 2 active site.

A major strategy for the development of a disease-modifying therapy against Alzheimer's disease is pharmacological intervention designed to reduce levels of beta-amyloid in the brain. Among various ways of reducing beta-amyloid production, the inhibition of beta-secretase (memapsin 2, BACE) is particularly attractive. Not only does beta-secretase initiates the amyloid cascade, it also is an aspartic protease, a class of proteases for which successful inhibitor drugs have been developed to treat AIDS patients. Extensive efforts in research and development of a beta-secretase inhibitor drug have taken place in many laboratories during the past few years. However, no drug candidate is currently in clinical trials. In spite of the lack of obvious success, much progress has been made to incorporate the drug-like properties in the evolution of better inhibitors. The inhibitors from more recent generations are indeed similar in characteristics to other protease inhibitor drugs. This progress permits optimism that development of clinical candidates of beta-secretase inhibitor drugs is a realistic goal.

Structure-based design and synthesis of a number of potent and selective memapsin 2 inhibitors are described. These inhibitors were designed based upon the X-ray structure of memapsin 2-bound inhibitor 3 that incorporates methylsulfonyl alanine as the P2-ligand and a substituted pyrazole as the P3-ligand. Of particular importance, we examined the ability of the substituted isophthalic acid amide derivative to mimic the key interactions in the S2-S3 regions of the enzyme active sites of 3-bound memapsin 2. We investigated various substituted phenylethyl, alpha-methylbenzyl, and oxazolylmethyl groups as the P3-ligands. A number of inhibitors exhibited very potent inhibitory activity against mempasin 2 and good selectivity against memapsin 1. Inhibitor 5d has shown low nanomolar enzyme inhibitory potency (Ki=1.1 nM) and very good cellular inhibitory activity (IC50=39 nM). Furthermore, in a preliminary study, inhibitor 5d has shown 30% reduction of Abeta40 production in transgenic mice after a single intraperitoneal administration (8 mg/kg). A protein-ligand X-ray crystal structure of 5d-bound memapsin 2 provided vital molecular insight that can serve as an important guide to further design of novel inhibitors.

Structure-based design, synthesis, and X-ray structure of protein-ligand complexes of memapsin 2 are described. The inhibitors are designed specifically to interact with S2- and S3-active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 6 has exhibited exceedingly potent inhibitory activity against memapsin 2 and selectivity over memapsin 1 (>3800-fold) and cathepsin D (>2500-fold). A protein-ligand crystal structure revealed cooperative interactions in the S2- and S3-active sites of memapsin 2. These interactions may serve as an important guide to design selectivity over memapsin 1 and cathepsin D.

Memapsin 2 (beta-secretase) is the membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP), leading to the production of amyloid-beta (Abeta), a major factor in the pathogenesis of Alzheimer's disease. The active site of memapsin 2 has been shown, with kinetic data and crystal structures, to bind to eight substrate residues (P(4)-P(4)'). We describe here that the addition of three substrate residues from P(7) to P(5) strongly influences the hydrolytic activity by memapsin 2 and these subsites prefer hydrophobic residues, especially tryptophan. A crystal structure of memapsin 2 complexed with a statine-based inhibitor spanning P(10)-P(4)' revealed the binding positions of P(5)-P(7) residues. Kinetic studies revealed that the addition of these substrate residues contributes to the decrease in K(m) and increase in k(cat) values, suggesting that these residues contribute to both substrate recognition and transition-state binding. The crystal structure of a new inhibitor, OM03-4 (K(i) = 0.03 nM), bound to memapsin 2 revealed the interaction of a tryptophan with the S(6) subsite of the protease.

A series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor (1, OM99-2 with eight residues) bound to memapsin 2. Ring size and substituent effects have been investigated. Cycloamide-urethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against human brain memapsin 2 (beta-secretase).

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.

We have previously reported structure-based design of memapsin 2 (beta-secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a "carrier peptide" penetrated the plasma membrane in cultured cells and inhibited the production of beta-amyloid (Abeta). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimer's mice (Tg2576) resulted in a significant decrease of Abeta level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Abeta reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.

Mempasin 2, a beta-secretase, is the membrane-anchored aspartic protease that initiates the cleavage of amyloid precursor protein leading to the production of beta-amyloid and the onset of Alzheimer's disease. Thus memapsin 2 is a major therapeutic target for the development of inhibitor drugs for the disease. Many biochemical tools, such as the specificity and crystal structure, have been established and have led to the design of potent and relatively small transition-state inhibitors. Although developing a clinically viable mempasin 2 inhibitor remains challenging, progress to date renders hope that memapsin 2 inhibitors may ultimately be useful for therapeutic reduction of beta-amyloid.

The discovery that beta-secretase is a membrane-anchored aspartic protease memapsin 2 has stimulated much interest in the design and testing of its inhibitors for the treatment of Alzheimer's disease. This article discusses the strategy for the development of such inhibitor drugs. Enzymology and structural determination tools have permitted the design of memapsin 2 inhibitors with high potency and in a size range possible for penetration of the blood-brain barrier. Transgenic Alzheimer's mice have been used to show that when memapsin 2 inhibitors are transported to the brain, they effectively reduce the production of amyloid beta. Although development of a clinical candidate of memapsin 2 inhibitor drug remains a very challenging undertaking, the progress so far lends some optimism for future prospects.

The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (Glu-Leu-Asp-LeuAla-Val-Glu-Phe, K(i) = 0.3 nM, the asterisk denotes the hydroxyethylene transition-state isostere) has been determined at 2.1 A resolution. Uniquely defined in the structure are the locations of S(3)' and S(4)' subsites, which were not identified in the previous structure of memapsin 2 in complex with the inhibitor OM99-2 (Glu-Val-Asn-LeuAla-Ala-Glu-Phe, K(i) = 1 nM). Different binding modes for the P(2) and P(4) side chains are also observed. These new structural elements are useful for the design of new inhibitors. The structural and kinetic data indicate that the replacement of the P(2)' alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P(3)' and P(4)'.

As beta-secretase, memapsin 2 cleaves amyloid-beta precursor protein, which leads ultimately to the onset of Alzheimer's disease. As such, memapsin 2 is an excellent target of inhibitor drugs for the treatment of this disease. Here we describe the tools for memapsin 2 inhibitor design that have been developed and results from the structure-based inhibitor design. Strategy for the design of memapsin 2 inhibitors with pharmaceutical potential is also discussed.

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.

Memapsin 1 is closely homologous to memapsin 2 (BACE), or beta-secretase, whose action on beta-amyloid precursor protein (APP) leads to the production of beta-amyloid (A beta) peptide and the progression of Alzheimer's disease. Memapsin 2 is a current target for the development of inhibitor drugs to treat Alzheimer's disease. Although memapsin 1 hydrolyzes the beta-secretase site of APP, it is not significantly present in the brain, and no direct evidence links it to Alzheimer's disease. We report here the residue specificity of eight memapsin 1 subsites. In substrate positions P(4), P(3), P(2), P(1), P(1)', P(2)', P(3)', and P(4)', the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe, and Trp, respectively, while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp, and Phe, respectively. Other less preferred residues can also be accommodated in these subsites of memapsin 1. Despite the broad specificity, these residue preferences are strikingly similar to those of human memapsin 2 [Turner et al. (2001) Biochemistry 40, 10001-10006] and thus pose a serious problem to the design of differentially selective inhibitors capable of inhibiting memapsin 2. This difficulty was confirmed by the finding that several potent memapsin 2 inhibitors effectively inhibited memapsin 1 as well. Several possible approaches to overcome this problem are discussed.

Chronic accumulation of beta-amyloid in the brain has been shown to result in complex molecular and cellular changes that accompany neurodegeneration in Alzheimer's disease (AD). In this study, we examined the expression of a newly identified beta-secretase, memapsin 2 (M2) or beta-site APP cleaving enzyme in deparaffinized sections from 10 AD patients and 10 aged matched controls and in frozen samples of parietal cortex from 11 AD and 8 controls. M2 is mainly expressed in neurons, with high levels in CA4 to CA2 regions and transentorhinal cortex and low or intermediate levels in CA1, subiculum, and granule cells of the dentate gyrus. The majority of AD brains showed an increase of M2 expression in the CA1, but a decrease in the transentorhinal cortex. A subset of controls and AD patients had high M2 expression in parietal neocortex. Double-staining revealed that senile plaques are not directly associated with the soma of M2-expressing neurons. Neurofibrillary tangles were associated with lower M2 expression in AD. These data indicate that beta-secretase M2 may not be straightforwardly involved in amyloid plaque formation in AD brain., (Copyright 2002 Elsevier Science (USA).)

Memapsin 2 (beta-secretase) is one of two proteases that cleave the beta-amyloid precursor protein (APP) to produce the 40-42 residue amyloid-beta peptide (Abeta) in the human brain, a key event in the progression of Alzheimer's disease. On the basis of the X-ray crystal structure of our lead inhibitor (2, OM99-2 with eight residues) bound to memapsin, we have reduced the molecular weight and designed potent memapsin inhibitors. Structure-based design and preliminary structure-activity studies have been presented.

Memapsin 2 is the protease known as beta-secretase whose action on beta-amyloid precursor protein leads to the production of the beta-amyloid (Abeta) peptide. Since the accumulation of Abeta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K(M) values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S(1) subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S(2), Asp > Asn > Met; S(3), Ile > Val > Leu; S(4), Glu > Gln > Asp; S(1)', Met > Glu > Gln > Ala; S(2)', Val > Ile > Ala; S(3)', Leu > Trp > Ala; S(4)', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K(M) value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S(2), Asp and Glu; S(3), Leu and Ile; S(2)', Val; and S(3)', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-LeuAla-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K(i) of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2 [Ghosh, A. K., et al. (2000) J. Am. Chem. Soc. 14, 3522-3523].

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease involved in the production of beta-amyloid peptide in Alzheimer's disease and is a major target for drug design. We determined the crystal structure of the protease domain of human memapsin 2 complexed to an eight-residue inhibitor at 1.9 angstrom resolution. The active site of memapsin 2 is more open and less hydrophobic than that of other human aspartic proteases. The subsite locations from S4 to S2' are well defined. A kink of the inhibitor chain at P2' and the change of chain direction of P3' and P4' may be mimicked to provide inhibitor selectivity.

Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosuppressive therapeutics. The secreted aspartic proteases (Saps) are known virulence factors of pernicious Candida species. The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence. Only one of these proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies. The other enzymes are present in low amounts in yeast culture and are difficult to purify. As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant zymogens and purified in large quantities. These proenzymes were autoactivated and purified as active proteases. The enzymic properties including the substrate specificities at the P(1) and P(1)' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites. At the P(1) site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged amino acids are also accommodated, especially by Sap2 and Sap3. The specificities at P(1)' are broader than at P(1) for all four enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic side chains are also accommodated at this site. Analysis of substrates with a hydrophobic amino acid in P(1)' reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp.

Autoantibodies binding the Sm B and B' peptides (B/B') are commonly associated with systemic lupus erythematosus in man and in MRL lpr/lpr mice. The linear antigenic regions of two anti-Sm B/B' murine monoclonal auto-antibodies have been mapped using overlapping octapeptides. Unique epitopes are identified by each antibody. Monoclonal KSm-5 recognizes the peptide, PPPGMRPP, which is repeated three times in the Sm B polypeptide. KSm 3 preferably binds to two similar, almost neighboring octapeptides, PPPGIRGP and PGIRGPPP. The two monoclonal antibodies do not cross react. These regions of Sm B/B' are major areas of antigenicity in human sera. Amino acid deletion and substitution in antigenic octapeptides show that binding to the KSm-5 epitope is lost with most modifications. Molecular dynamic modelling suggests that when PPPGMRPP is substituted in the sixth position arginine, KSm/5 binding may be associated with a shared peptide backbone structure rather than charge or hydrophobicity of the substituted amino acid. In contrast, binding of KSm-3 to PPPGIRGP is abolished when the sixth position arginine is substituted by any other amino acid. Substitution at arginine and modelling experiments, therefore, suggest very different mech-anisms of binding. Autoantibodies may bind quite different features of similar peptide structures., (Copyright 1999 Academic Press.)

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens including DNA, ribosomal P, Ro (SS-A), La (SS-B), and the spliceosome. Etiology is suspected to involve genetic and environmental factors. Evidence of genetic involvement includes: associations with HLA-DR3, HLA-DR2, Fcgamma receptors (FcgammaR) IIA and IIIA, and hereditary complement component deficiencies, as well as familial aggregation, monozygotic twin concordance >20%, lambdas > 10, purported linkage at 1q41-42, and inbred mouse strains that consistently develop lupus. We have completed a genome scan in 94 extended multiplex pedigrees by using model-based linkage analysis. Potential [log10 of the odds for linkage (lod) > 2.0] SLE loci have been identified at chromosomes 1q41, 1q23, and 11q14-23 in African-Americans; 14q11, 4p15, 11q25, 2q32, 19q13, 6q26-27, and 12p12-11 in European-Americans; and 1q23, 13q32, 20q13, and 1q31 in all pedigrees combined. An effect for the FcgammaRIIA candidate polymorphism) at 1q23 (lod = 3.37 in African-Americans) is syntenic with linkage in a murine model of lupus. Sib-pair and multipoint nonparametric analyses also support linkage (P < 0.05) at nine loci detected by using two-point lod score analysis (lod > 2.0). Our results are consistent with the presumed complexity of genetic susceptibility to SLE and illustrate racial origin is likely to influence the specific nature of these genetic effects.

Basic amino acids Arg, Lys, and His in the Cys2His2 zinc fingers of transcription factor IIIA (TFIIIA) potentially have important roles in factor binding to the extended internal control region (ICR) of the 5S ribosomal gene. Conserved and non-conserved basic residues in the N-terminal fingers I, II, III and the more C-terminal fingers V and IX were analyzed by site-directed mutagenesis and DNase I protection in order to assess their individual requirement in the DNA-binding mechanism. In the DNA recognition helix of finger II, the conserved Arg at position 62 (N-terminal side of the first zinc-coordinating histidine) was changed to a Leu or Gln. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gene ICR. When His-58 (non-conserved basic residue with DNA-binding potential in the same helical region) was changed to a Gln, the mutated protein was able to protect the ICR from DNase I digestion. Therefore, Arg-62 is individually required for TFIIIA binding over the entire ICR whereas His-58 is not. Fingers V and IX have conserved Arg residues in positions identical to Arg-62 in finger II (Arg-154 in finger V and Arg-271 in finger IX). When these residues were changed to Leu and Ile respectively, TFIIIA-dependent DNase I protection was observed along the entire 5S gene ICR. These results indicate differing DNA-binding mechanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-terminal finger I, the conserved Lys at position 11 outside the recognition helix and a conserved hydrophobic Trp at position 28 within the helix were changed to an Ala and Ser respectively. The K11A change inhibited TFIIIA-dependent DNase I protection to a much greater extent than the W28S change.

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin). Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep. The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains. Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme. However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate. In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)