Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis

Mapping Inhibitor Binding Modes on an Active Cysteine Protease via Nuclear Magnetic Resonance Spectroscopy

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [¹⁵N]­Cys, [¹⁵N]­His, and [¹³C]­Met labeling was performed to quickly assess cruzain–ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme–inhibitor interactions to facilitate lead compound identification and subsequent structural studies

Clinical outcomes and proximal junctional failure in adult spinal deformity patients corrected to normative alignment versus functional alignment.

OBJECTIVE: The objective of this study was to explore the rate of proximal junctional failure (PJF) and functional outcomes of normative alignment goals compared with alignment targets based on age-appropriate physical function. METHODS: Baseline relationships between age, pelvic incidence (PI), and a component of the T1 pelvic angle (TPA) within the fusion were analyzed in adult spinal deformity (ASD) patients and compared with those of asymptomatic patients. Linear regression modeling was used to determine alignment based on PI and age in asymptomatic patients (normative alignment), and in ASD patients, alignment corresponding to age-appropriate functional status (functional alignment). A cohort of 288 ASD patients was split into two groups based on whether the patient was closer to their normative or functional alignment goal at their 6-week postoperative radiographic follow-up. The rates of proximal junctional kyphosis (PJK) and PJF were determined for each cohort. RESULTS: In the 288 ASD patients included in this pre- to postoperative analysis, there was no difference in baseline alignment or health-related quality of life (HRQOL) between the normative alignment and functional alignment groups. At 6 weeks, patients with normative alignment had a smaller TPA (4.45° vs 14.1°) and PI minus lumbar lordosis (-7.24° vs 7.4°) (both p \u3c 0.0001) and higher PJK (40% vs 27.2%, p = 0.03) and PJF (17% vs 6.8%, p = 0.008) rates than patients with functional alignment. CONCLUSIONS: Correction in ASD patients to normative alignment resulted in higher rates of PJK and PJF without improvements in HRQOL. Correction in ASD patients to functional alignment that mirrors the physical function of their age-matched asymptomatic peers is recommended

Clinical outcomes and proximal junctional failure in adult spinal deformity patients corrected to normative alignment versus functional alignment

OBJECTIVE: The objective of this study was to explore the rate of proximal junctional failure (PJF) and functional outcomes of normative alignment goals compared with alignment targets based on age-appropriate physical function. METHODS: Baseline relationships between age, pelvic incidence (PI), and a component of the T1 pelvic angle (TPA) within the fusion were analyzed in adult spinal deformity (ASD) patients and compared with those of asymptomatic patients. Linear regression modeling was used to determine alignment based on PI and age in asymptomatic patients (normative alignment), and in ASD patients, alignment corresponding to age-appropriate functional status (functional alignment). A cohort of 288 ASD patients was split into two groups based on whether the patient was closer to their normative or functional alignment goal at their 6-week postoperative radiographic follow-up. The rates of proximal junctional kyphosis (PJK) and PJF were determined for each cohort. RESULTS: In the 288 ASD patients included in this pre- to postoperative analysis, there was no difference in baseline alignment or health-related quality of life (HRQOL) between the normative alignment and functional alignment groups. At 6 weeks, patients with normative alignment had a smaller TPA (4.45° vs 14.1°) and PI minus lumbar lordosis (-7.24° vs 7.4°) (both p \u3c 0.0001) and higher PJK (40% vs 27.2%, p = 0.03) and PJF (17% vs 6.8%, p = 0.008) rates than patients with functional alignment. CONCLUSIONS: Correction in ASD patients to normative alignment resulted in higher rates of PJK and PJF without improvements in HRQOL. Correction in ASD patients to functional alignment that mirrors the physical function of their age-matched asymptomatic peers is recommended

Mapping Inhibitor Binding Modes on an Active Cysteine Protease via Nuclear Magnetic Resonance Spectroscopy

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (&gt;30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies