RESUMEN
Under healthy conditions, the cornea is an avascular structure which allows for transparency and optimal visual acuity. Its avascular nature is maintained by a balance of proangiogenic and antiangiogenic factors. An imbalance of these factors can result in abnormal blood vessel proliferation into the cornea. This corneal neovascularization (CoNV) can stem from a variety of insults including hypoxia and ocular surface inflammation caused by trauma, infection, chemical burns, and immunological diseases. CoNV threatens corneal transparency, resulting in permanent vision loss. Mainstay treatments of CoNV have partial efficacy and associated side effects, revealing the need for novel treatments. Numerous natural products and synthetic small molecules have shown potential in preclinical studies in vivo as antiangiogenic therapies for CoNV. Such small molecules include synthetic inhibitors of the vascular endothelial growth factor (VEGF) receptor and other tyrosine kinases, plus repurposed antimicrobials, as well as natural source-derived flavonoid and non-flavonoid phytochemicals, immunosuppressants, vitamins, and histone deacetylase inhibitors. They induce antiangiogenic and anti-inflammatory effects through inhibition of VEGF, NF-κB, and other growth factor receptor pathways. Here, we review the potential of small molecules, both synthetics and natural products, targeting these and other molecular mechanisms, as antiangiogenic agents in the treatment of CoNV.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Neovascularización de la Córnea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Neovascularización de la Córnea/patología , Humanos , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Multiple proteases in a system hydrolyze target substrates, but recent evidence indicates that some proteases will degrade other proteases as well. Cathepsin S hydrolysis of cathepsin K is one such example. These interactions may be uni- or bi-directional and change the expected kinetics. To explore potential protease-on-protease interactions in silico, a program was developed for users to input two proteases: (1) the protease-ase that hydrolyzes (2) the substrate, protease. This program identifies putative sites on the substrate protease highly susceptible to cleavage by the protease-ase, using a sliding-window approach that scores amino acid sequences by their preference in the protease-ase active site, culled from MEROPS database. We call this PACMANS, Protease-Ase Cleavage from MEROPS ANalyzed Specificities, and test and validate this algorithm with cathepsins S and K. PACMANS cumulative likelihood scoring identified L253 and V171 as sites on cathepsin K subject to cathepsin S hydrolysis. Mutations made at these locations were tested to block hydrolysis and validate PACMANS predictions. L253A and L253V cathepsin K mutants significantly reduced cathepsin S hydrolysis, validating PACMANS unbiased identification of these sites. Interfamilial protease interactions between cathepsin S and MMP-2 or MMP-9 were tested after predictions by PACMANS, confirming its utility for these systems as well. PACMANS is unique compared to other putative site cleavage programs by allowing users to define the proteases of interest and target, and can also be employed for non-protease substrate proteins, as well as short peptide sequences.
Asunto(s)
Algoritmos , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Proteolisis , Programas InformáticosRESUMEN
To achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation.
Asunto(s)
Anafase , Cromosomas Humanos/metabolismo , Metafase , Anafase/efectos de los fármacos , Cromátides/metabolismo , Células HeLa , Humanos , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Metafase/efectos de los fármacos , Modelos Biológicos , Movimiento , Ácido Ocadaico/farmacología , Fosforilación/efectos de los fármacosRESUMEN
Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume that class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery, as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, shape, elongation, division, sporulation (SEDS)-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria.
RESUMEN
Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.
Asunto(s)
Actinas/metabolismo , Hipocampo/metabolismo , Modelos Biológicos , Imagen Molecular/métodos , Neuronas/citología , Neuronas/metabolismo , Animales , Teorema de Bayes , Células Cultivadas , Simulación por Computador , Femenino , Células HeLa , Hipocampo/citología , Humanos , Cadenas de Markov , Ratones , MicroARNs/metabolismo , Microscopía Fluorescente/métodos , Modelos Estadísticos , Reconocimiento de Normas Patrones Automatizadas/métodos , Transporte de Proteínas/fisiología , Programas InformáticosRESUMEN
Cathepsins S and K are potent mammalian proteases secreted into the extracellular space and have been implicated in elastin and collagen degradation in diseases such as atherosclerosis and osteoporosis. Studies of individual cathepsins hydrolyzing elastin or collagen have provided insight into their binding and kinetics, but cooperative or synergistic activity between cathepsins K and S is less described. Using fluorogenic substrate assays, Western blotting, cathepsin zymography, and computational analyses, we uncovered cathepsin cannibalism, a novel mechanism by which cathepsins degrade each other as well as the substrate, with cathepsin S predominantly degrading cathepsin K. As a consequence of these proteolytic interactions, a reduction in total hydrolysis of elastin and type I collagen was measured compared with computationally predicted values derived from individual cathepsin assays. Furthermore, type I collagen was preserved from hydrolysis when a 10-fold ratio of cathepsin S cannibalized the highly collagenolytic cathepsin K, preventing its activity. Elastin was not preserved due to strong elastinolytic ability of both enzymes. Together, these results provide new insight into the combined proteolytic activities of cathepsins toward substrates and each other and present kinetic models to consider for more accurate predictions and descriptions of these systems.
Asunto(s)
Catepsina K/química , Catepsinas/química , Colágeno Tipo I/química , Modelos Químicos , Proteolisis , Catepsina K/genética , Catepsina K/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Humanos , HidrólisisRESUMEN
Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3-8 pN to 6-12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.
Asunto(s)
Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Materiales Biocompatibles , Ingeniería Biomédica , Endocitosis , Matriz Extracelular/metabolismo , Heparina/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Inmovilizadas/metabolismo , Pinzas Ópticas , Fosforilación , Estabilidad Proteica , Solubilidad , Propiedades de Superficie , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.