Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.     

ACID-INDUCED FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been known for some time that morning glory filaments elongate in response to increases in concentration of gibberellins (Murakami, 1973) and decreases in ethylene production (Koning and Raab, in press), but many other aspects of their growth have remained unstudied. In the present work, the possible role of gibberellin-stimulated proton efflux in filament growth was examined. Although applied gibberellins stimulated extensive filament growth in vitro and the pH of the incubating medium became acidified during growth, gibberellin also induced growth in media buffered at alkaline pH values. Acidic buffers alone elicited only a very small amount of growth. Fusicoccin, a potent stimulator of proton efflux, initially stimulated the rate of filament growth but elicited only a small increment of growth. In fact, continued presence of fusicoccin poisoned sustained gibberellin-induced growth. Vanadate ions, believed to inhibit proton efflux, had little effect upon gibberellin-induced growth except at extremely high concentrations. Based upon these results, it appears that the acid-induced component of growth stimulation by gibberellin is relatively minor in Ipomoea filaments. These results are quite different from those reported for filament elongation in Gaillardia (Koning, 1983a).     

ROLE OF GROWTH SUBSTANCES IN THE FILAMENT GROWTH OF FUCHSIA HYBRIDA CV “BRILLIANT”

Filaments of Fuchsia hybrida cv “Brilliant” double in length within 24 hr after bud opening. Filament growth characterized by fresh wt increase and cell elongation was significantly inhibited in vitro by l-aminocyclopropane-l-carboxylic acid (ACC) but was not promoted by any growth regulator tested. Ions of Co²⁺ blocked the inhibitive effects of ACC in vitro suggesting that ethylene produced from ACC is the growth inhibiting substance. Ethylene levels surrounding the filaments within the closed bud decreased during development, and premature opening of the sepals which released the ethylene into the atmosphere resulted in rapid filament growth. The ACC levels were found to be much higher in the anthers than the filaments. This suggests that ethylene produced from floral organs other than filaments regulates filament elongation in Fuchsia. This is the first report of filament growth which cannot be promoted by application of growth regulators but which is inhibited by ethylene.     

Genetical genomics in humans and model organisms

Genetical genomics has been proposed to map loci controlling gene-expression differences (eQTLs) that might underlie functional trait variation. We briefly review the studies in model species and conclude that, although they successfully demonstrate the utility of genetical genomics, they are too limited to unlock the full potential of this approach and some results should be interpreted with caution. We subsequently elaborate on two recent studies that use this approach in humans. The many differences between these studies complicate meaningful comparisons between them. A joint analysis of the two experiments offers some scope for more powerful genetical genomics.     

Experimental evaluation of the power balance model of speed skating.

Prediction of speed skating performance with a power balance model requires assumptions about the kinetics of energy production, skating efficiency, and skating technique. The purpose of this study was to evaluate these parameters during competitive imitations for the purpose of improving model predictions. Elite speed skaters (n = 8) performed races and submaximal efficiency tests. External power output (P(o)) was calculated from movement analysis and aerodynamic models and ice friction measurements. Aerobic kinetics was calculated from breath-by-breath oxygen uptake (Vo(2)). Aerobic power (P(aer)) was calculated from measured skating efficiency. Anaerobic power (P(an)) kinetics was determined by subtracting P(aer) from P(o). We found gross skating efficiency to be 15.8% (1.8%). In the 1,500-m event, the kinetics of P(an) was characterized by a first-order system as P(an) = 88 + 556e(-0.0494t) (in W, where t is time). The rate constant for the increase in P(aer) was -0.153 s(-1), the time delay was 8.7 s, and the peak P(aer) was 234 W; P(aer) was equal to 234[1 - e(-0.153(t-8.7))] (in W). Skating position changed with preextension knee angle increasing and trunk angle decreasing throughout the event. We concluded the pattern of P(aer) to be quite similar to that reported during other competitive imitations, with the exception that the increase in P(aer) was more rapid. The pattern of P(an) does not appear to fit an "all-out" pattern, with near zero values during the last portion of the event, as assumed in our previous model (De Koning JJ, de Groot G, and van Ingen Schenau GJ. J Biomech 25: 573-580, 1992). Skating position changed in ways different from those assumed in our previous model. In addition to allowing improved predictions, the results demonstrate the importance of observations in unique subjects to the process of model construction.     

Hospital and community ampicillin-resistant Enterococcus faecium are evolutionarily closely linked but have diversified through niche adaptation

Background

Ampicillin-resistant Enterococcus faecium (ARE) has emerged as a nosocomial pathogen. Here, we quantified ARE carriage in different community sources and determined genetic relatedness with hospital ARE.

Methods and Results

ARE was recovered from rectal swabs of 24 of 79 (30%) dogs, 11 of 85 (13%) cats and 0 of 42 horses and from 3 of 40 (8%) faecal samples of non-hospitalized humans receiving amoxicillin. Multi-locus Sequence Typing revealed 21 sequence types (STs), including 5 STs frequently associated with hospital-acquired infections. Genes previously found to be enriched in hospital ARE, such as IS16, orf903, orf905, orf907, were highly prevalent in community ARE (≥79%), while genes with a proposed role in pathogenesis, such as esp, hyl and ecbA, were found rarely (≤5%) in community isolates. Comparative genome analysis of 2 representative dog isolates revealed that the dog strain of ST192 was evolutionarily closely linked to two previously sequenced hospital ARE, but had, based on gene content, more genes in common with the other, evolutionarily more distantly related, dog strain (ST266).

Conclusion

ARE were detected in dogs, cats and sporadically in healthy humans, with evolutionary linkage to hospital ARE. Yet, their accessory genome has diversified, probably as a result of niche adaptation.     

NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.