Nitration of cathepsin D enhances its proteolytic activity during mammary gland remodelling after lactation.
Proteomic studies in the mammary gland of control lactating and weaned rats have shown that there is an increased pattern of nitrated proteins during weaning when compared with controls. Here we report the novel finding that cathepsin D is nitrated during weaning. The expression and protein levels of this enzyme are increased after 8 h of litter removal and this up-regulation declines 5 days after weaning. However, there is a marked delay in cathepsin D activity since it does not increase until 2 days post-weaning and remains high thereafter. In order to find out whether nitration of cathepsin D regulates its activity, iNOS (inducible nitric oxide synthase)(-/-) mice were used. The expression and protein levels of this enzyme were similar to WT (wild-type) animals, but the proteolytic activity was significantly reduced during weaning in knockout compared to WT mice. in vitro treatment of recombinant human cathepsin D or lactating mammary gland homogenates with relatively low concentrations of peroxynitrite enhances the nitration as well as specific activity of this enzyme. Using MS, it has been shown that the residue Tyr168 was nitrated. All of these results show that protein nitration during weaning might be a signalling pathway involved in mammary gland remodelling.
Zaragozá
Rosa
R
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, E-46010 Valencia, Spain.
Torres
Luis
L
García
Concha
C
Eroles
Pilar
P
Corrales
Fernando
F
Bosch
Ana
A
Lluch
Ana
A
García-Trevijano
Elena R
ER
Viña
Juan R
JR
D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D015151:Immunoblotting; D047468:Immunoprecipitation; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D009566:Nitrates / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000502:physiology; D011247:Pregnancy; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014886:Weaning
D009566:Nitrates; D052247:Nitric Oxide Synthase Type II; D002402:Cathepsin D