Critical Cleavage Research Articles

Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

Proteolytic activation of the human epithelial sodium channel by trypsin and trypsin IV involves distinct cleavage sites (1181.2)

Proteolytic channel activation is a unique feature of the epithelial sodium channel (ENaC) but is not yet fully understood. The serine protease trypsin is known to activate ENaC in vitro but may not be relevant in vivo. In this study, we wanted to investigate whether the trypsin‐like serine protease trypsin IV, known to be expressed in several epithelial tissues, can activate human ENaC. Moreover, using site‐directed mutagenesis we wanted to identify functionally relevant cleavage sites in the γ‐subunit of the channel. In the Xenopus laevis oocyte expression system, we monitored the proteolytic activation of ENaC currents and the appearance of γ‐ENaC cleavage products at the cell surface. We demonstrated that trypsin IV can stimulate ENaC and that this activation involves a critical cleavage site (K189) in the extracellular domain of the γ‐subunit. Moreover, we showed that channel activation by trypsin was prevented by mutating three putative trypsin cleavage sites (K168; K170; R172) in addition to mutating previously described prostasin (RKRK178), plasmin (K189), and neutrophil elastase (V182;V193) sites. In conclusion, we demonstrated for the first time that trypsin IV can activate ENaC and that trypsin and trypsin IV use specific cleavage sites to achieve channel activation. Preferential cleavage sites may provide a mechanism for differential ENaC regulation by tissue‐specific proteases.Grant Funding Source: Supported by grants of the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (S.H., M.K.)

Fracture toughness of a welded super duplex stainless steel

Fracture toughness testing was conducted on standard single-edge notched bend bar specimens of base and weld metal. The material was the SAF 2906 super duplex stainless steel. The aim was to evaluate the susceptibility for brittle failure at sub-zero temperatures for the base and weld metal. The base metal was tested between −103 and −60°C and was evaluated according to the crack-tip opening displacement method. The fracture event at and below −80°C can be described as ductile until critical cleavage initiation occurs, which caused unstable failure of the specimen. The welding method used was submerged arc welding with a 7wt% nickel filler metal. The welded specimens were post-weld heat treated (PWHT) at 1100°C for 20min and then quenched. Energy-dispersive X-ray spectroscopy analysis showed that during PWHT substitutional element partitioning occurred which resulted in decreased nickel content in the ferrite. The PWHT weld metal specimens were tested at −72°C. The fracture sequence was critical cleavage fracture initiation after minor crack-tip blunting and ductile fracture.

Quantitative Investigation of Brittle Out-of-plane Fracture in X70 Pipeline Steel

Brittle out-of-plane cracking (by delamination or brittle tilted fracture) affects the impact toughness of pipeline steels. It has been investigated on notched tensile specimens using a local approach to fracture. Smooth and notched bars taken along four directions (including the short transverse direction, ND) have been tested in tension at temperatures between 20°C and -196°C. Delamination partly results from the lower value of the critical cleavage stress along ND, linked to the microtexture anisotropy, but also from the presence of ductile microcracks acting as stress concentrators triggering fracture along the rolling plane. Brittle tilted fracture was associated to a relatively lower value of the critical cleavage stress in that plane, but prior delamination was necessary to trigger it. The relevance of a macroscopic critical stress criterion for delamination is finally discussed.

Delamination of pipeline steels: determination of an anisotropic cleavage criterion

Cleavage fracture anisotropy has been studied on a ferrite-bainite low alloy pipeline steel plate. Mechanical tests have been performed on smooth and notched bars taken along the three principal directions of the plate at temperatures between 20 °C and −196 °C, and analyzed by finite element calculations with an anisotropic plasticity model. The fracture mode of specimens tested along the normal direction differs from that of specimens taken along the two other directions, together with a lower critical cleavage stress. This difference seems to be related to microtexture anisotropy and might explain the sensitivity of this steel to delamination at low temperatures.

Critical Stress for Cleavage Fracture in Continuously Cooled Medium Carbon V-microalloyed Steel

Cleavage fracture of the medium carbon V-microalloyed steel with structure consisting of acicular ferrite, pearlite and grain boundary ferrite has been investigated by means of four-point bending of the notched samples at -196°C. It was found that cleavage fracture initiation has not been related to the coarse second phase particles cracking. Calculated values of the effective surface energy of 49J/m2 and critical cleavage fracture stress normalized by yield stress of 1.84 are in agreement with the results for the steels with ferrite-pearlite and bainite structures.

Propagation and arrest of cleavage cracks in a nuclear pressure vessel steel

The safety of nuclear structures is crucial while the service time of nuclear power stations is planned to be extended up to 60years. Initiation stage of cracks is still considered as a key issue, but more and more component integrity analyses investigate the crack arrest possibility. This study deals with physical mechanisms of cleavage crack propagation and numerical computations related to brittle fracture. Experiments using standard measuring techniques and a high-speed framing camera system, as well as Scanning Electron Microscope fracture surface analyses were carried out on thin CT specimens made of 16MND5 PWR vessel steel. The elastic–viscoplastic behavior of the ferritic steel has been studied and taken into account in numerical simulations. The eXtended Finite Element Method (X-FEM) is used in CAST3M finite element analysis software to model crack propagation. Numerical computations combine a local non-linear dynamic approach and a fracture criterion based on critical cleavage stress, whereas current standards in the nuclear field use a global static approach to fracture to depict crack initiation and arrest. The links of the criterion with temperature and strain rate are considered thanks to experiments, SEM fractographies and 2D computations in order to get a robust physical model which can be effective for model-based predictions of industrial structures.

Energetic and Structural Basis for Activation of the Epithelial Sodium Channel by Matriptase

Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors, and other proteins synthesized as inactive precursors. Serine proteases make up one of the largest and most conserved families of endopeptidases involved in diverse cellular activities, including wound healing, blood coagulation, and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle's syndrome are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for the enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that has previously not been identified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. On the basis of our results, we propose a dynamics-driven "anglerfish" mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase than by furin.

Mechanisms of human neutrophil elastase-catalysed inactivation of factor VIII(a)

Mechanisms of inflammation and coagulation are linked through various pathways. Human neutrophil elastase (HNE), can bind to activated platelets, might be localised on platelet membranes that provide negatively-charged phospholipid essential for the optimum function of tenase complex. In this study, we examined the effect of HNE on factor (F)VIII. FVIII activity was rapidly diminished in the presence of HNE and was undetectable within 10 minutes. The inactivation rate was ~8-fold greater than that of activated protein C (APC). This time-dependent inactivation was moderately affected by von Willebrand factor. HNE proteolysed the heavy chain (HCh) of FVIII into two terminal products, A11-358 and A2375-708, by limited proteolysis at Val358, Val374, and Val708. Cleavage at Val708 was much slower than that at Val358 in the >90-kDa A1-A2-B compared to the 90-kDa A1-A2. The 80-kDa light chain (LCh) was proteolysed to 75-kDa product by cleavage at Val1670. HNE-catalysed FVIIIa inactivation was markedly slower than that of native FVIII (by ~25-fold), due to delayed cleavage at Val708 in FVIIIa. The inactivation rate mediated by HNE was ~8-fold lower than that by APC. Cleavages at Val358 and Val708 were regulated by the presence of LCh and HCh, respectively. In conclusion, HNE-catalysed FVIII inactivation was associated with the limited-proteolysis that led to A11-358, A2375-708, and A3-C1-C21671-2332, and subsequently to critical cleavage at Val708. HNE-related FVIII(a) reaction might play a role in inactivation of HNE-induced coagulation process, and appeared to depend on the amounts of inactivated FVIII and active FVIIIa which is predominantly resistant to HNE inactivation.

Microstructure and Cleavage Resistance of High Strength Steels

The relationship between microstructure and cleavage resistance in quenched and tempered high strength bainitic and martensitic steels is investigated by means of Charpy-V three-point bending tests, uniaxial tensile test on unnotched specimens and EBSD. Steels under investigation are low/medium carbon (C=0.10%-0.40%) steels with yield strength in the range YS=500-1000 MPa. Results show that the tensile strength and the cleavage resistance of Q&T steels appear to be controlled by different structural parameters and not, as in the case of polygonal ferritic steels, by the same structural unit. In particular, yield strength is controlled by the mean subgrain size, whereas the structural unit controlling the critical cleavage stress is the covariant (bainitic or martensitic) packet, whose size is slightly lower than the average unit crack path (UCP). The critical stage in the fracture process appears to be the propagation of a Griffith crack from one packet to another, and the resistance offered by high-angle boundaries is approximately the same as that of low-C steels with bainitic or polygonal ferrite microstructure.

First-principles modeling of hardness in transition-metal diborides

Based on recent experiments, the diborides ${\text{OsB}}_{2}$ and ${\text{ReB}}_{2}$ were proposed to be ultraincompressible and superhard materials. By application of an ab initio density-functional theory approach we investigate the elastic and cleavage fracture properties of the borides $M{\text{B}}_{2}$ ($M=\text{ }\text{Hf}$, Ta, W, Re, Os, and Ir). We derive a direct correlation between the lowest calculated critical cleavage stress and the experimental (micro)hardness. By calculating the critical shear stress and estimating the possibility of dislocation emission we can justify the prediction that ${\text{ReB}}_{2}$ is indeed a superhard material.

Effect of microstructure on cleavage resistance of high-strength quenched and tempered steels

The relationship between microstructure and cleavage resistance in quenched and tempered high-strength bainitic and martensitic steels is investigated by means of Charpy-V tests, uniaxial tensile test on unnotched specimens and electron back scattered diffraction (EBSD). Steels under investigation are low/medium carbon (C = 0.10–0.40%) steels with yield strength in the range YS = 500–1000 MPa. Results show that the tensile strength and the cleavage resistance of quenched and tempered (Q&T) steels appear to be controlled by different structural parameters and not, as in the case of polygonal ferritic steels, by the same structural unit. In particular, yield strength is controlled by the mean subgrain size, whereas the structural unit controlling the critical cleavage stress is the covariant (bainitic or martensitic) packet, whose size is slightly lower than the average unit crack path (UCP). The critical stage in the fracture process appears to be the propagation of a Griffith crack from one packet to another, and the resistance offered by high-angle boundaries is approximately the same as that of low-C steels with bainitic or polygonal ferrite microstructure.

First-principles study of the ideal cleavage fracture of Cr 2Nb microalloyed by X (Al, Ni, Co, Ti)

The structural properties and elastic constants of the pure Cr 2Nb, the ideal cleavage fracture properties of the Cr 2Nb with the elements X (Al, Ni, Co and Ti) and site preference of the elements X were investigated using the first-principles method. Our calculated lattice parameters and elastic constants of the pure Cr 2Nb are in agreement with the experimental data. Moreover, our calculated results show that the elements X (Al, Ni and Co) occupy the Cr site, and Ti the Nb site in Cr 2Nb. The cleavage energies G c and critical cleavage stress σ c with and without the additive elements in the cleavage planes were calculated. The results demonstrate that the elements X (Al, Ni, Co and Ti) could be used to improve the cleavage properties of Cr 2Nb. Finally, the electronic mechanism behind the effects of the additive elements on the ideal cleavage properties of Cr 2Nb was investigated by calculating the electronic structure.

Dependence of critical cleavage stresses as a function of orientation and temperature in single crystals of Fe-18% Cr-14% Ni-2% Mo austenitic stainless steel containing hydrogen

The effect of electrolytic hydrogen saturation on the critical cleavage stress τ cr was studied as a function of test temperature, duration of hydrogen saturation, and orientation of the tension axis in single crystals of Fe-18% Cr-14% Ni-2% Mo austenitic stainless steel.

Using the X-FEM method to model the dynamic propagation and arrest of cleavage cracks in ferritic steel

Although initiation criteria have been the subject of many publications, the phenomena associated with the propagation and arrest of brittle cracks have not. To be able to predict the dynamic behaviour of cleavage cracks, we made a series of experiments and associated numerical studies. Tests of crack propagation and arrest were carried out on specimens of two different geometries (Compact Tension and compression ring) made of the 16MND5 ferritic steel of which French nuclear reactor vessels are constructed. The elastic-viscoplastic behaviour of this material was characterised and its nature was taken into account in the numerical simulations. The eXtended Finite Element Method (X-FEM) was developed in CAST3M finite element analysis software to enable fine, effective modelling of crack propagation. Propagation models based on principal stress were studied and it was found that critical cleavage stress depended on loading rate. The use of criteria calibrated for Compact Tension specimens gave excellent results in predictive calculations for similar specimens, and also for compression rings in both mode I and mixed-mode. The speed and path of crack predicted with the numerical simulations were in close agreement with the experimental results.

Huntingtin's critical cleavage

The early pathogenic events leading to neurodegeneration in Huntington disease are not clear. A recent paper shows that mutating a caspase-6 cleavage site in the huntingtin protein is sufficient to prevent pathogenesis.

Substrate specificity of inner membrane peptidase in yeast mitochondria

The inner membrane protease (IMP) cleaves intra-organelle sorting peptides from precursor proteins in mitochondria of the yeast Saccharomyces cerevisiae. An unusual feature of the IMP is the presence of two catalytic subunits, Imp1p and Imp2p, which recognize distinct substrate sets even though both enzymes belong to the same protease family. This nonoverlapping substrate specificity was hypothesized to result from the recognition of distinct residues at the P'1 position (also termed +1 position) in the protease substrates. Here, we constructed an extensive series of mutations to obtain a profile of the critical cleavage site residues in IMP substrates and conclude that Imp1p, and not Imp2p, recognizes specific P'1 residues. In addition to its specificity for P'1 residues, Imp1p also shows substrate specificity for the P3 (-3) position. In contrast, Imp2p recognizes the P1 (-1) position and the P3 position. Based on this new understanding of IMP substrate specificity, we conducted a survey for candidate IMP substrates in mammalian mitochondria and found consensus Imp2p cleavage sites in mammalian precursors to cytochrome c1 and glycerol-3-phosphate (G-3-P) dehydrogenase. Presence of a putative Imp2p cleavage site in G-3-P dehydrogenase was surprising, as its yeast ortholog contains an Imp1p cleavage site. To address this issue experimentally, we performed the first co-expression of mammalian IMP with proposed mammalian IMP precursors in yeast and show that murine precursors to cytochrome c1 and G-3-P dehydrogenase are cleaved by murine Imp2p. These results suggest, surprisingly, G-3-P dehydrogenase has switched from Imp1p in yeast to Imp2p in mammals.

Failure analysis of cold drawn prestressing steel wires subjected to stress corrosion cracking

This paper analyzes the stress corrosion performance of cold drawn prestressing steels with different degrees of cold drawing, in the two cases of pure stress corrosion cracking and fracture by hydrogen embrittlement. Results show a progressively anisotropic stress corrosion behaviour with cold drawing, and this anisotropy could be induced by the microstructural orientation. Consequences for engineering design are derived on the basis of the calculation of the transition stress intensity factor (from the stress corrosion subcritical crack growth to the critical cleavage propagation). While in situations of pure stress corrosion cracking such a value is even higher than the fracture toughness of the material in air (due to crack tip blunting), in the case of hydrogen embrittlement there is a marked reduction of stress intensity factor value, which indicates that these steels are highly susceptible to this phenomenon.

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake

The origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa-factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P. textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at approximately 280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

Specimen size effects on ductile–brittle transition temperature in Charpy impact testing

One key issue for small specimen test techniques is to clarify specimen size effects on test results. In consideration of size effects on determining the ductile-to-brittle transition temperature (DBTT) in Charpy impact testing, a method to evaluate the plastic constraint loss for differently sized Charpy V-notch (CVN) specimens is proposed and applied to a ferritic–martensitic steel, 2WFK, developed by JNC. In the method, a constraint factor, α, that is an index of the plastic constraint is defined as α=σ ∗/σ y ∗ . Here, σ ∗ is the critical cleavage fracture stress which is a material constant and σ y ∗ is the uniaxial yield stress at the DBTT at the strain rate generated in the Charpy impact test. The procedures for evaluating each of σ ∗ and σ y ∗ are described and a result of σ ∗ and σ y ∗ , thus the value of α, is presented for different types of miniaturized and full-sized CVN specimens of 2WFK.