A molecular map of titin/connectin elasticity reveals two different mechanisms acting in series

In the I-band of skeletal muscle sarcomeres, the elastic region of titin consists of immunoglobulin (Ig) domains, and non-modular regions rich in proline, hydrophobic, and charged residues (PEVK). Using immunoelectron microscopy with sequence-assigned monoclonal antibodies, we demonstrate that extension of the Ig regions in M. psoas occurs largely at sarcomere lengths between 2 and 2.8 micron, decreasing in slope towards higher lengths. The Ig domains do not unfold. Above 2.6 micron, length changes are increasingly due to the PEVK-rich regions. We therefore propose that rubber-like properties of the PEVK-rich regions are mainly contributing to skeletal titin elasticity.     

Binding of the N-terminal 63 kDa portion of connectin/titin to alpha-actinin as revealed by the yeast two-hybrid system

BACKGROUND: Corneal blood staining may occur as a serious complication of persisting hyphema. In our two cases the corneal blood staining is a result of direct bleeding in the corneal stroma. HISTORY AND SIGNS: A female patient presented with a corneal blood staining through rupture of reopened vessels in interstitial keratitis of congenital syphilis after physical effort with high blood pressure. The second patient presented with a blood staining caused by a vessel errosion after corneal ulceration. THERAPY AND OUTCOME: The clearing of the blood staining is thought to be a result of the phagocytic action of the keratocytes and from a diffusion of hemoglobin into the conjunctival circulation and the anterior chamber (2). The therapeutic efforts are directed toward prevention of corneal blood staining. At the first sign of microscopic blood staining of the cornea a surgical evacuation of hyphema is necessary. In our two cases there wasn't a hyphema, so it was only possible to treated the corneal ulcer, entropion and systemic hypertension. CONCLUSION: The clinician has to wait for a spontaneous clearing, although it may take 2 or 3 years or more. In this case a penetrating keratoplasty is indicated.     

Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.     

Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.     

Inhibition of tumour cell invasion by protease inhibitors: correlation with the protease profile

The invasive potential of eight established human tumour cell lines of different origin has been studied in the Matrigel assay. Between 25% and 70% of the cells migrated through the Matrigel layer within 24 h, indicating that invasiveness varies with the cell type. Semiquantitative measurements of the proteases MMP-2 and MMP-9, and cathepsins B and L were performed in these cell lines and the cell culture media. High invasive potential was found in those cell lines expressing high levels of cathepsins B and L or matrix metal proteases (MMP), either alone or in combination. Overexpression of one of these enzymes is enough to explain a high invasive potential of a cell line. Selective protease inhibitors at 10 nM concentration in the culture medium were used to inhibit the migration of tumour cells in the Matrigel assay. The MMP inhibitor Batimastat reduced the invasive potential of all cell lines studied independently of the MMP expression. The effect of cysteine protease inhibitors was strongly correlated with the protease profile of the tumour cell line. Our findings support the hypothesis of a very complex activation cascade of matrix-degrading proteolytic enzymes and they underline the need to analyse the protease profile of any tumour before beginning an antiproteolytic tumour treatment.     

Protein protease inhibitors in insects and comparison with mammalian inhibitors

1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results.     

Effects of electrical stimulation and postmortem storage on changes in titin, nebulin, desmin, troponin-T, and muscle ultrastructure in Bos indicus crossbred cattle

The effects of electrical stimulation (ES) on degradation of titin, nebulin, desmin, and troponin-T (TN-T) and on structural changes in the longissimus muscle (LM) from Brahman x Simmental (B x S) cattle (Bos indicus cross) were determined. The left side of seven B x S beef carcasses was stimulated (200 V, 20 Hz) within 1 h of death, and the right side was the nonstimulated (NS) control. Myofibrils for SDS-PAGE and samples for transmission electron microscopy were prepared from the LM at 0, 1, 3, 7, 14, and 28 d postmortem (PM). The SDS-PAGE results showed that the T1 band of titin was absent by 7 d in two animals, by 14 d in four animals, and by 28 d in one animal in both NS and ES samples. By SDS-PAGE, intact nebulin was gone by 7 d in two animals and by 14 d in five animals, but in blots, nebulin decreased by 7 d and was absent by 14 d in both NS and ES samples. The desmin band could still be seen as a light band at 28 d in Western blots of both NS and ES samples. A decrease in TN-T and a concomitant increase in the 30-kDa polypeptide were observed in both NS and ES samples. Western blots with a monoclonal antibody to TN-T confirmed that TN-T decreased at similar rates in NS and ES samples but showed that the 30-kDa polypeptide was more heavily labeled in ES samples from 7 to 28 d. Contraction nodes were present in O-d ES samples and were still observed in 28-d ES samples. Narrow, intermediate, and wide I-band fractures were seen earlier and at a greater frequency in ES than in NS samples. Overall, ES had no detectable effect on titin, nebulin, desmin, or TN-T degradation but accelerated the appearance and enhanced the frequency of three types of I-band fractures in the LM from Bos indicus crossbred cattle.     

Fibrinolysis with des-kringle derivatives of plasmin and its modulation by plasma protease inhibitors

Quantitative characterization of the interaction of des-kringle1-5-plasmin (microplasmin) with fibrin(ogen) and plasma protease inhibitors may serve as a tool for further evaluation of the role of kringle domains in the regulation of fibrinolysis. Comparison of fibrin(ogen) degradation products yielded by plasmin, miniplasmin (des-kringle1-4-plasmin), microplasmin, and trypsin on SDS gel electrophoresis indicates that the differences in the enzyme structure result in different rates of product formation, whereas the products of the four proteases are very similar in molecular weight. Kinetic parameters show that plasmin is the most efficient enzyme in fibrinogen degradation, and the kcat/KM ratio decreases in parallel with the loss of the kringle domains. The catalytic sites of the four proteases have similar affinities for fibrin (KM values between 0.12 and 0.21 microM). Trypsin has the highest catalytic constant for fibrin digestion (kcat = 0.47 s-1), and among plasmins with different kringle structures, the loss of kringle5 results in a markedly lower catalytic rate constant (kcat = 0.0076 s-1 for microplasmin vs 0.048 s-1 for miniplasmin and 0.064 s-1 for plasmin). In addition, microplasmin is inactivated by plasmin inhibitor (k" = 3.9 x 10(5) M-1 s-1) and antithrombin (k" = 1.4 x 10(3) M-1 s-1) and the rate of inactivation decreases in the presence of fibrin(ogen). Heparin (250 nM) accelerates the inactivation of microplasmin by antithrombin (k" = 10.5 x 10(3) M-1 s-1 ), whereas that by plasmin inhibitor is not affected (k" = 4.2 x 10(5) M-1 s-1).     

Peptidomimetic inhibitors of the human cytomegalovirus protease

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.     

The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease

This paper is a review of the literature on the possible association between osteoporosis and oral bone loss, with an emphasis on radiological studies. Such an association was first suggested in 1960. Subsequent histomorphometric and microradiographic studies showed that after the age of 50 there was a marked increase in the cortical porosity of the mandible, with this increase being greater in the alveolar bone than the mandibular body; and that with this increase in porosity, there was a concomitant decrease in bone mass, which appeared to be more pronounced in females than in males, with the loss in bone mineral content estimated to be 1.5% per year in females and 0.9% in males. These studies also demonstrated a considerable amount of variation in the amounts of cortical and trabecular bone within and among individuals. Subsequent clinical studies reported associations between the bone densities of jaws and (1) metacarpals, (2) forearm bones, (3) vertebrae and (4) femurs. These studies indicated that women had lower mandibular bone mineral content (BMC) than men and that age-related loss of bone was more pronounced in women after the age of 50 years than in men of the same age, as was the case for the rest of the body. It was suggested that systemic factors responsible for osteoporotic bone loss may combine with local factors (periodontal diseases) to increase rates of periodontal alveolar bone loss. Although not all studies found associations between osteoporosis and oral bone loss, the conclusion of this review is that such an association exists; yet additional longitudinal investigations are needed to confirm this, and before the implications of this association could be fully utilized in clinical dentistry, inexpensive methods must be developed for sensitive and specific measures of oral bone loss.     

Calpain inhibitors and serine protease inhibitors can produce apoptosis in HL-60 cells

Recent investigations indicate that proteolysis is an important event in generation of the apoptosis phenotype. Although various proteases have been suggested to be candidates for this proteolysis, the results from different laboratories are inconsistent. In the present studies, HL-60 cells were treated with cycloheximide to investigate proteases involved in apoptosis. The calpain inhibitors benzyloxycarbonyl-Leu-Leu-Tyr diazomethylketone and acetyl-Leu-Leu-Nle aldehyde were not capable of preventing apoptosis induced by cycloheximide. In the absence of cycloheximide, these two inhibitors could initiate apoptosis in HL-60 cells. The thiol protease inhibitor benzyloxycarbonyl-Leu-Val-Gly diazomethylketone neither prevented nor produced apoptosis. The serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and tosyl-Phe chloromethylketone (TPCK) also induced apoptosis in the absence of cycloheximide. On the other hand, the latter two inhibitors decreased cycloheximide-induced apoptosis, assessed either by cell morphologic changes or DNA ladder generation. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and iodoacetamide, inactivators of interleukin 1beta-converting enzyme (ICE)-like proteases, did not produce apoptosis and inhibited the induction of apoptosis by cycloheximide, calpain inhibitors, or serine protease inhibitors. These results are consistent with the ICE-like proteases having a central role in proteolysis during apoptosis, while calpain-like proteases and the serine proteases sensitive to DCI or TPCK are not required for generation of the apoptosis phenotype in HL-60 cells.     

Automated docking and the search for HIV protease inhibitors

This article will discuss the motivations, technologies, and future directions of computational automated docking in the context of the structure-based rational design of HIV-1 protease inhibitors. Docking simulations are widely used for screening of compound libraries to identify new drug leads, employing a simple model for rapid testing of thousands of compounds. Docking simulations are also useful for lead enhancement, using more detailed models to analyze the atomic interactions between inhibitors and target macromolecules. Major advances have been reported in the development of empirical force fields, which now allow assessment of relative binding strength and drug specificity, and extensions of automated docking techniques allow de novo drug design.     

Involvement of protease inhibitors in staphylokinase-induced fibrin-specific fibrinolysis

We compared the fibrinolytic properties of recombinant staphylokinase (SAK), a fibrin-specific plasminogen activator, with those of streptokinase and tissue-type plasminogen activator (t-PA) by means of the amidolytic method. We also investigated the involvement of alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin in SAK-induced fibrin-specific fibrinolysis. Both SAK and t-PA activated plasminogen efficiently in the presence of fibrin in human plasma. Although t-PA activated plasminogen dependently on fibrin in the reconstituted plasma system, SAK activated plasminogen independently of fibrin without alpha 2-plasmin inhibitor (alpha 2-antiplasmin, alpha 2-PI). These findings suggest that fibrin and alpha 2-PI play important roles in plasminogen activation by SAK but not by t-PA. Furthermore, protease inhibitors such as alpha 2-PI, alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin inhibited plasminogen activation by SAK and the inhibitory actions of these protease inhibitors disappeared in the presence of fibrin. This shows that alpha 2-macroglobulin, C1-inactivator and alpha 1-antitrypsin, other than alpha 2-PI, contribute to the fibrin-specificity of SAK.     

Design and synthesis of novel conformationally restricted HIV protease inhibitors

A set of HIV protease inhibitors represented by compound 2 has previously been described. Structural and conformational analysis of this compound suggested that conformational restriction of the P1/P2 portion of the molecule could lead to a novel set of potent protease inhibitors. Thus, probe compounds 3-7 were designed, synthesized, and found to be potent inhibitors of HIV protease.     

Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.     

Molecular recognition of cyclic urea HIV-1 protease inhibitors

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.     

Substrates and inhibitors of human T-cell leukemia virus type I protease

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.