Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins

Abstract

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.

Chromatin remodeling during sea urchin early development: molecular determinants for pronuclei formation and transcriptional activation

“…As the activity of the SpH cysteine protease is maximal at pH 8.0, its role in the progression from step II to step III gains further support. Moreover, the presence of an inactive form of the SpH-cysteine protease in unfertilized eggs is in agreement with a male pronucleus formation process independent from protein synthesis after fertilization (Krystal and Poccia, 1979;Imschenetzky et al, 1991aImschenetzky et al, , 1997.…”

“…Hence, the degradation of sperm histones appears as a necessary step in male chromatin remodeling after fertilization. A novel 60 kDa cysteine protease, first identified in our laboratory, was found present as a proenzyme in unfertilized sea urchin eggs and activated shortly after fertilization (Imschenetzky et al, 1997). At pH 8.0, which is the condition found post-insemination in sea urchin eggs (Whitaker and Steinhardt, 1985), this enzyme specifically degrades all five SpH, leaving the maternally derived CS histone variants intact.…”

Characterization of proteolytic activities during intestinal regeneration of the sea cucumber, Holothuria glaberrima

“…In addition, inhibitors of cysteine proteases delay the phase S of the cell cycle (Goulet et al, 2007) and affect the embryonic cell cycle progression in the sea urchin (Morin et al, 2008). Moreover, in vivo experiments with E64d showed inhibition of sperm histones degradation which is necessary for pronuclear formation during fertilization process (Imschenetzky et al, 1997). Therefore, we cannot exclude a possible contribution of cysteine proteases (cathepsin L) on cell division during intestinal regeneration.…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…A candidate for the maternal kinase is Cdk2, because it colocalizes with the male pronucleus after fertilization (Schnackenberg and Marzluff, 2002;reviewed in Imschenetzky et al, 2003). Once their attachment has been loosened, sperm-specific histones are removed from sperm chromatin by a maternal sperm-nucleosome disassembly activity (SDNA) and then degraded by a maternal protease (Imschenetzky et al, 1997;Iribarren et al, 2008).…”