RESUMEN

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Asunto(s)

Pared Celular/metabolismo , Citrus/microbiología , Glucanos/metabolismo , Glicósido Hidrolasas/metabolismo , Factores de Virulencia/genética , Xanthomonas/metabolismo , Xilanos/metabolismo , Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Activación Transcripcional , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismo , Xanthomonas/genética , Xanthomonas/patogenicidad

RESUMEN

BACKGROUND: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. METHODS: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. RESULTS: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. CONCLUSION: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

RESUMEN

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Asunto(s)

Animales , Venenos de Serpiente , Antivenenos , Bothrops , Serina Proteasas , Péptidos

RESUMEN

Background: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom’s, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

RESUMEN

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Asunto(s)

Animales , Venenos de Serpiente , Antivenenos , Bothrops , Serina Proteasas , Péptidos

RESUMEN

Diabetes mellitus accelerates vascular calcification (VC) and increases the risk of end-stage renal disease (ESRD). Nevertheless, the impact of VC in renal disease progression in type 2 diabetes mellitus (T2DM) is poorly understood. We addressed the effect of VC and mechanisms involved in renal dysfunction in a murine model of insulin resistance and obesity (ob/ob), comparing with their healthy littermates (C57BL/6). We analyzed VC and renal function in both mouse strains after challenging them with Vitamin D3 (VitD3). Although VitD3 similarly increased serum calcium and induced bone disease in both strains, 24-hour urine volume and creatinine pronouncedly decreased only in ob/ob mice. Moreover, ob/ob increased urinary albumin/creatinine ratio (ACR), indicating kidney dysfunction. In parallel, ob/ob developed extensive intrarenal VC after VitD3. Coincidently with increased intrarenal vascular mineralization, our results demonstrated that Bone Morphogenetic Protein-2 (BMP-2) was highly expressed in these arteries exclusively in ob/ob. These data depict a greater susceptibility of ob/ob mice to develop renal disease after VitD3 in comparison to paired C57BL/6. In conclusion, this study unfolds novel mechanisms of progressive renal dysfunction in diabetes mellitus (DM) after VitD3 in vivo associated with increased intrarenal VC and highlights possible harmful effects of long-term supplementation of VitD3 in this population.

Asunto(s)

Colecalciferol/farmacología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Suplementos Dietéticos , Resistencia a la Insulina , Enfermedades Renales/patología , Calcificación Vascular/complicaciones , Animales , Hormonas y Agentes Reguladores de Calcio/farmacología , Enfermedades Renales/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/fisiopatología

RESUMEN

Objective- We hypothesized that ob/ob mice develop expansive vascular remodeling associated with calcification. Approach and Results- We quantified and investigated mechanisms of vascular remodeling and vascular calcification in ob/ob mice after vitamin D3(VD) stimulation or PBS (control), compared with C57BL/6 mice. Both ob/ob (OBVD [VD-treated ob/ob mice]) and C57BL/6 (C57VD [VD-treated C57BL/6 mice]) received 8×103 IU/day of intraperitoneal VD for 14 days. Control ob/ob (OBCT [PBS-treated ob/ob mice]) and C57BL/6 (C57CT [PBS-treated C57BL/6 mice]) received intraperitoneal PBS for 14 days. Hypervitaminosis D increased the external and internal elastic length in aortae from OBVD, resulting in increased total vascular area and lumen vascular area, respectively, which characterizes expansive vascular remodeling. OBVD decreased the aortic wall thickness, resulting in hypotrophic vascular remodeling. We demonstrated increased collagen deposition, elastolysis, and calcification in aortae from OBVD. Our results showed a positive correlation between expansive vascular remodeling and vascular calcification in OBVD. We demonstrated increased serum calcium levels, augmented Bmp (bone morphogenetic protein)-2 and osteochondrogenic proteins expression in OBVD aortae. Furthermore, aortae from OBVD increased oxidative stress, coincidently with augmented in situ MMP (matrix metalloproteinase) activity and exhibited no VDR (VD receptor) inhibition after VD. Conclusions- Our data provide evidence that obese and insulin-resistant mice (ob/ob) developed expansive hypotrophic vascular remodeling correlated directly with increased vascular calcification after chronic VD stimulation. Positive hypotrophic vascular remodeling and vascular calcification in this mouse model is possibly mediated by the convergence of absence VDR downregulation after VD stimulation, increased reactive oxygen species generation, and MMP activation.

Asunto(s)

Colecalciferol/farmacología , Resistencia a la Insulina , Obesidad/complicaciones , Calcificación Vascular/inducido químicamente , Remodelación Vascular/efectos de los fármacos , Animales , Calcio/sangre , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Receptores de Calcitriol/fisiología , Remodelación Vascular/fisiología

RESUMEN

We report that two fractions containing proteins from rat hepatocyte nuclei, obtained by nondenaturing gel electrophoresis, were able to bind iron and ATP, and to hydrolyze ATP. Electroelution of these two active fractions followed by SDS-PAGE analysis showed an identical protein pattern, each one containing four proteins in a range of 62-80 kDa. Phosphorylated protein bands were also detected in acid gel and disappeared after treatment with hydroxylamine/acetate or KOH, and upon chasing with cold ATP. A proteoliposome system, made by the incorporation of these partially purified protein fractions into phosphatidylcholine vesicles, carried out Fe(3+)-citrate uptake in a Mg(2+)-ATP-dependent way; Fe(3+) accumulation increased with time reaching a plateau in 30 min. Iron uptake was not supported by AMP-PNP, was partially inhibited by orthovanadate and was not affected by a mix of specific inhibitors of known ATPases. These results support our previous hypothesis that a putative nuclear membrane Fe(3+)-ATPase is involved in nuclear iron homeostasis.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Compuestos Férricos/metabolismo , Proteolípidos/metabolismo , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Animales , Núcleo Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Hepatocitos/ultraestructura , Membrana Nuclear/enzimología , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Fosforilación , Ratas

RESUMEN

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.

Asunto(s)

Glicoproteínas/química , Glicoproteínas/fisiología , Neuraminidasa/química , Neuraminidasa/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/patogenicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Glicoproteínas/genética , Humanos , Queratina-18/química , Queratina-18/metabolismo , Sistema de Señalización de MAP Quinasas , Neuraminidasa/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Virulencia