Heterozygous CAPN3 missense variants causing autosomal-dominant calpainopathy in seven unrelated families.

AIMS: Recessive variants in CAPN3 gene are the cause of the commonest form of autosomal recessive limb girdle muscle dystrophy. However, two distinct in-frame deletions in CAPN3 (NM_000070.3:c.643_663del21 and c.598_621del15) and more recently, Gly445Arg and Arg572Pro substitutions have been linked to autosomal dominant (AD) forms of calpainopathy. We report 21 affected individuals from seven unrelated families presenting with an autosomal dominant form of muscular dystrophy associated with five different heterozygous missense variants in CAPN. METHODS: We have used massively parallel gene sequencing (MPS) to determine the genetic basis of a dominant form of limb girdle muscular dystrophy in affected individuals from seven unrelated families. RESULTS: The c.700G> A, [p.(Gly234Arg)], c.1327T> C [p.(Ser443Pro], c.1333G> A [p.(Gly445Arg)], c.1661A> C [p.(Tyr554Ser)] and c.1706T> C [p.(Phe569Ser)] CAPN3 variants were identified. Affected individuals presented in young adulthood with progressive proximal and axial weakness, waddling walking and scapular winging or with isolated hyperCKaemia. Muscle imaging showed fatty replacement of paraspinal muscles, variable degrees of involvement of the gluteal muscles, and the posterior compartment of the thigh and minor changes at the mid-leg level. Muscle biopsies revealed mild myopathic changes. Western blot analysis revealed a clear reduction in calpain 3 in skeletal muscle relative to controls. Protein modelling of these variants on the predicted structure of calpain 3 revealed that all variants are located in proximity to the calmodulin-binding site and are predicted to interfere with proteolytic activation. CONCLUSIONS: We expand the genotypic spectrum of CAPN3-associated muscular dystrophy due to autosomal dominant missense variants.

Characterisation of hypertensive heart disease: pathological insights from a sudden cardiac death cohort to inform clinical practice.

Hypertensive heart disease refers to changes in the myocardium that result from hypertension. The relationship between hypertensive heart disease and sudden cardiac death is well established, but there are few pathological studies. We examined the clinical and pathological features of hypertensive heart disease in sudden cardiac death victims from a national cardiovascular pathology registry. We investigated 5239 cases of sudden cardiac death between 1994 and 2018. Hearts were examined by two expert cardiac pathologists. Diagnostic criteria included history of hypertension, increased heart weight and left ventricular wall thickness in the absence of other causes. Collagen was quantified using picrosirius red staining and imaging software. Of 75 sudden cardiac death cases due to hypertensive heart disease (age at death: 54 ± 16 years; 56% males), 56 (75%) reported no prior cardiac symptoms. Thirty-four (45%) recorded a BMI ≥ 30. Only two (2.7%) had hypertensive heart disease diagnosed antemortem. Four (5%) were diagnosed clinically with hypertrophic cardiomyopathy, but lacked myocyte disarray at autopsy. All hearts showed concentric left ventricular hypertrophy and myocyte hypertrophy. Fibrosis was identified microscopically in 59 cases (81%). The posterior left ventricular wall showed the greatest increase in the percentage of collagen in hypertensive diseased hearts compared to controls (25.2% vs 17.9%, p = 0.034). Most sudden deaths due to hypertensive heart disease occur without prior cardiac symptoms; thus, clinical risk stratification is challenging. Hypertensive heart disease can be misdiagnosed in life as hypertrophic cardiomyopathy which has major implications for relatives. Pathologists require a history of hypertension and histology for a definitive diagnosis of hypertensive heart disease.

Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin.

BACKGROUND: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. OBJECTIVE: Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.

Identification of domains influencing assembly and ion channel properties in alpha 7 nicotinic receptor and 5-HT3 receptor subunit chimaeras.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT(3)Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR alpha 7 subunit and the 5-HT(3) receptor (3A) subunit. EXPERIMENTAL APPROACH: A series of subunit chimaeras containing alpha 7 and 5-HT(3A) subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. KEY RESULTS: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on alpha 7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some alpha 7/5-HT(3A) chimaeras. CONCLUSIONS AND IMPLICATIONS: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties.

Reactive site mutants of recombinant protein C inhibitor.

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, alpha 1-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353-->P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.

Increased expression of the mRNA for hormone-sensitive lipase in adipose tissue of cancer patients.

The expression of genes coding for regulatory enzymes involved in the uptake, synthesis and mobilisation of lipid was measured in adipose tissue of cancer patients. Total RNA was isolated from subcutaneous adipose tissue of control and cancer patients and the various mRNAs measured by Northern blot analysis. The total lipoprotein lipase enzymic activity and the relative levels of the mRNAs for lipoprotein lipase and for fatty acid synthase were not significantly different between cancer patients and control patients. However, there was a significant two-fold increase in the relative level of mRNA for hormone-sensitive lipase (HSL) in adipose tissue of cancer patients compared with control patients. The cancer patients also exhibited a two-fold elevation in serum triacylglycerol levels and serum free fatty acid levels. There was a significant correlation between the serum free fatty acid level and expression of HSL mRNA in the adipose tissue. The serum levels of insulin and tumour necrosis factor-alpha were not different between cancer and control patients. The results suggest that at least one of the mechanisms for depletion of lipid from adipose tissue in cancer patients operates at the level of increased expression of mRNA of the lipolytic regulatory enzyme, hormone-sensitive lipase.

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a-specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.

Pig apolipoprotein R: a new member of the short consensus repeat family of proteins.

Apolipoprotein R (apoR) is a 23-kDa protein found on very low-density lipoprotein (VLDL), on chylomicrons, and in the d > 1.21 g/mL fraction of pig plasma. The plasma concentration of apoR is 5.1 micrograms/mL, with 11.5% of apoR found on VLDL. In vitro, apoR can transfer from the d > 1.21 g/mL infranatant onto artificial lipid emulsions or human chylomicrons but not onto human VLDL. An apoR cDNA was isolated from a pig liver lambda gt11 expression library. DNA sequence analysis of the apoR cDNA revealed 67% identity with the 3'-terminal region of human C4b-binding protein alpha-chain cDNA (C4BP alpha). C4BP alpha is a 70-kDa glycoprotein that regulates both the coagulation and the complement cascades. In plasma, C4BP alpha exists as disulfide-linked multimers consisting of seven C4BP alpha chains and a single C4BP beta chain. Like C4BP, apoR forms high molecular weight disulfide-linked complexes in plasma. However, unlike C4BP alpha, apoR complexes do not appear to contain C4BP beta. ApoR mRNA was detected in pig liver, spleen, lung, bone marrow, and lymph node, but was absent in intestine and white blood cells. This distribution is consistent with the production of apoR in terminally differentiated macrophages but not in blood monocytes. ApoR mRNA was not detected in RNA isolated from human liver or lung. ApoR may be a lipoprotein-borne regulator of either the coagulation or the complement cascades.

Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit.

Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) alpha7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of alpha7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned alpha7 cDNA. In contrast, elevated levels of alpha-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "alpha7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous alpha7 subunit. However, by expression of an epitope-tagged alpha7 subunit, it has been possible to show that the elevation in surface alpha-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded alpha7. The cell-specific misfolding of the neuronal nAChR alpha7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homooligomeric serotonin receptor 5-HT3 subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.

Thrombin enhances monocyte secretion of tumor necrosis factor and interleukin-1 beta by two distinct mechanisms.

Thrombosis and disseminated intravascular coagulation (DIC) are common complications of infections. Abnormal activation of coagulation is due in part of expression of tissue factor on intravascular cells in response to cytokines, including interleukin-1 beta (IL1 beta ) and tumor necrosis factor (TNF). Both TNF and IL1 beta are thought to play significant roles in producing the pathologic manifestations of sepsis. Therefore, we examined the effects of thrombin on TNF and IL1 beta secretion of monocytes, and the ability of monocyte products to promote tissue factor expression by endothelial cells. Human monocytes were treated with thrombin or a thrombin receptor agonist peptide (SFLLRN), and/or bacterial lipopolysaccharide (LPS). The agonists were removed, and monocytes cultured 18 hours. The monocyte-conditioned supernatants were assayed for TNF and IL1 beta antigen, and for their ability to induce tissue factor expression on human umbilical vein endothelial cells and the Ea.hy endothelial cell line. Thrombin alone did not promote monocyte TNF or IL-1 beta secretion. However, thrombin enhanced LPS-induced TNF and IL1 secretion. Supernatants from monocytes exposed to LPS plus thrombin promoted greater tissue factor expression on endothelial cells than supernatants from those treated with LPS only. SFLLRN did not increase TNF secretion in response to LPS, but did enhance LPS-induced IL1 beta secretion and tissue factor-inducing activity. Neither SFLLRN nor active thrombin augmented the level of mRNA for TNF above that induced by LPS alone. However, both increased the LPS-induced level of IL1 beta message. Thus, thrombin enhanced LPS-induced TNF and IL1 beta secretion by monocytes. Unexpectedly, the effects on these two cytokines were mediated by different mechanisms. Enhancement of LPS-induced IL1 beta secretion was largely mediated via the tethered ligand type thrombin receptor and correlated with an increase in the steady state level of mRNA. By contrast, enhanced TNF required proteolytically active thrombin, but was not mediated by the tethered ligand receptor. These data demonstrate that physiologically relevant amounts of thrombin can synergize with endotoxin to stimulate monokine release. Thrombin could thereby play a role in the complex network of mediators involved in the pathophysiology of sepsis. We speculate that limiting thrombin activity during DIC could be a beneficial adjunct in the management of sepsis.

Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit.

Heterologous expression of the neuronal nicotinic acetylcholine receptor alpha8 subunit in cultured mammalian cell lines has revealed that the correct folding of this protein is dependent on the host cell type. The alpha8 subunit, which is able to form homo-oligomeric ion channels when expressed in Xenopus oocytes, could be detected in all transfected cell lines by both immunoprecipitation and immunofluorescence microscopy with a monoclonal antibody that recognises a linear epitope. In contrast, the alpha8 subunit could be detected in some but not in all transfected cell lines with a monoclonal antibody that recognises a conformation-sensitive epitope or by nicotinic radioligand binding. It is interesting that although correctly folded alpha8 protein could be detected in transfected rat pituitary (GH4C1) cells, only misfolded alpha8 protein could be detected in a large subpopulation of transfectants (transient or clonal stable isolates). We have also found that the protein encoded by a chimaeric cDNA (constructed from the N-terminal region of alpha8 and the C-terminal domain of the serotonin 5-HT3 receptor subunit) is expressed efficiently, and in a conformation that binds alpha-bungarotoxin, in all cell types examined. These results, together with previous expression studies with the homo-oligomeric alpha7 subunit and hetero-oligomeric nicotinic receptor subunit combinations, suggest that the cell-specific folding described here is a phenomenon that may be characteristic of homo-oligomeric nicotinic receptors.

Assessment of the interaction between urokinase and reactive site mutants of protein C inhibitor.

Urokinase-type plasminogen activator (uPA) is a serine protease involved in pericellular proteolysis and tumor cell metastasis via plasmin-mediated degradation of extracellular matrix proteins. Plasma uPA is inhibited by the serine protease inhibitor protein C inhibitor (PCI) by the insertion of PCI's reactive site loop into the active site of the protease. To better understand the structural aspects of this inhibition, 15 reactive-site mutants of recombinant PCI (rPCI) were assayed for differences in uPA inhibition. These assays revealed that substitutions at the P1 Arg354 and P3 Thr352 sites of rPCI were detrimental to inhibitory activity, while P3' Arg357 mutations had little effect upon the inhibition rate. However, replacement of the P2 Phe353 with small residues like Ala and Gly increased the effectiveness of rPCI three- to four fold. To explain these altered rates of inhibition, a computer-derived molecular model of uPA was generated and docked to a model of PCI to simulate complex formation. The changes made by mutagenesis were then recreated in the model of uPA-PCI. In accordance with the kinetic data, the poor performance of P3 variants is primarily attributable to charge repulsion, while alleviation of steric hindrance at P2 produces the observed increase in uPA inhibition. In the model, residues at P3' interact with PCI rather than uPA, consistent with P3' variants demonstrating that little variation from wild-type activity. Ultimately, this combination of mutagenesis and molecular modeling will further refine our understanding of the interaction between PCI and uPA.

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs.

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb5. Lpb5 pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb5 pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.

Protein C inhibitor is a potent inhibitor of the thrombin-thrombomodulin complex.

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI approximately equal to 140-fold (k2 = 2.4 x 10(6) in the presence of TM compared to 1.7 x 10(4) M-1 S-1 in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI approximately equal to 59-fold (k2 = 1.0 x 10(6) M-1 S-1). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe353 with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k2 = 6.3 x 10(5) M-1 S-1), but TM 4-6 enhances inhibition by this mutant approximately equal to 9-fold (k2 = 5.8 x 10(6) M-1 S-1) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.

Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity.

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.

Intermolecular interactions between protein C inhibitor and coagulation proteases.

Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.

Construction of recombinant adeno-associated virus vector containing the rat preproinsulin II gene.

We have investigated a possible delivery system for the rat preproinsulin II gene (rI2) utilising a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC12BI (ATCC) was purified and digested with restriction enzymes SepI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rI2). Subsequently, the RSV-rI2 gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI2 recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine kinase promoter-driven gene for neomycin resistance (neoR). The cell line 293 (ATCC) was then cotransfected with pLP-1 and helper plasmid pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI2, was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed by Southern blot analysis using the rI2 gene probe derived from the original plasmid. The final titer of 1.25 x 10(9) particles/ ml was determined by DNA slot blots using pLP-1 as the standard, HPAC cells were infected with vLP-1 (termed HPAC/rI2). Integration of the rI2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI2 gene by PCR. Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin II gene. Successful transduction and stable integration of rI2 into HPAC was achieved. Production of insulin by HPAC/rI2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.

Distal regulatory elements control MRF4 gene expression in early and late myogenic cell populations.

MRF4 is a muscle-specific transcription factor that belongs to a family of basic helix-loop-helix proteins known as the myogenic regulatory factors (MRFs). In vitro studies have shown that expression of the MRF4 gene is controlled by a proximal promoter element (-336 to +71) that binds the muscle-specific transcription factors MEF2 and myogenin to activate transcription. To examine further the regulatory elements necessary for endogenous MRF4 gene expression during development, transgenic mice were generated that contained either a proximal MRF4 promoter-LacZ reporter gene (-336 MRF4-nLacZ) or a MRF4-LacZ reporter gene containing 8.5 kb of 5' flanking sequence (-8500 MRF4-nLacZ). Characterization of individual transgenic mouse lines throughout development revealed that expression of both transgenes is restricted to skeletal muscle tissue. However, unlike previous in vitro data, the proximal promoter transgene exhibits only limited transcriptional activity at all developmental time points, whereas the -8500 MRF4-nLacZ lines fully recapitulate the later developmental expression patterns and exhibit transcription in myotomal cells during somitic differentiation. Tissue culture analysis of myogenic cells isolated from E12.5, E16.5, and adults confirmed that the -8500 MRF4-nLacZ transgene is expressed in greater than 90% of the myotubes for all myogenic populations. These results indicate that 8.5 kb of MRF4 5' flanking sequence contains all the regulatory elements necessary for late MRF4 expression and that at least some of these elements lie upstream of the -336 proximal promoter. It is also likely that distant upstream regulatory sequences control early somitic MRF4 expression. These findings, coupled with previous in vitro studies, suggest that the early and late developmental expression patterns of the MRF4 gene are controlled by distinct sets of regulatory elements.