Microinjection of an antibody against the cysteine‐protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle

Puchi, Marcia; Quiñones, Karin; Concha, Carolina; Iribarren, Claudio; Bustos, Paula; Morín, Violeta; Genevière, Anne Marie; Imschenetzky, María

doi:10.1002/jcb.20800

Cited by 13 publications

(21 citation statements)

References 29 publications

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“…In functional terms, this enzyme is involved in histones metabolism after fertilization [Imschenetzky et al, 1993] and also in cell cycle control. Its inhibition blocks SpH degradation, then provokes the arrest of the initial cell cycle and ultimately aborts early development [Concha et al, 2005b;Puchi et al, 2006]. Interestingly, a murine nuclear variant of cathepsin L was also postulated to be involved in cell cycle control.…”

Section: Discussionmentioning

confidence: 96%

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

Iribarren

Morín

Puchi

et al. 2007

J of Cellular Biochemistry

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We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.

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Section: Discussionmentioning

confidence: 96%

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

Iribarren

Morín

Puchi

et al. 2007

J of Cellular Biochemistry

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“…Of the antibody-based protease inhibitors that have been reported, [5][6][7][8][9][10][11] most work by interfering with protein-protein interaction sites rather than interacting with the active site of the enzyme. Previously, we used a phage-displayed single-chain antibody library to develop potent and specific inhibitors of membrane type serine protease 1 (MT-SP1/matriptase), but the molecular details of the inhibitory mechanism were unclear.…”

Section: Introductionmentioning

confidence: 99%

Structure of an Fab–Protease Complex Reveals a Highly Specific Non-canonical Mechanism of Inhibition

Farady

Egea

Schneider

et al. 2008

Journal of Molecular Biology

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SummaryThe vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition, but due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be greatly aided by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.

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“…A third approach has been to mature specific protease inhibitors on other natural protein scaffolds, such as ankyrin repeats 15 or antibodies. To this point, the characterized protease antibody inhibitors have been monoclonal antibodies raised from hybridomas, and have tended towards two types of inhibitors; inhibitors that interfere with multimerization (and thus activation) of the protease 16,17,18 , and inhibitors that bind to loops and protein-protein interaction sites 19, 20, 21; 22 and occlude substrate binding, instead of interfering with the catalytic machinery of the enzyme, and ensuring complete inhibition 23 .…”

Section: Introductionmentioning

confidence: 99%

The Mechanism of Inhibition of Antibody-based Inhibitors of Membrane-type Serine Protease 1 (MT-SP1)

Farady¹,

Sun²,

Darragh³

et al. 2007

Journal of Molecular Biology

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SummaryThe mechanisms of inhibition of two novel scFv antibody inhibitors of the serine protease MT-SP1/ matriptase reveal the basis of their potency and specificity. Kinetic experiments characterize the inhibitors as extremely potent inhibitors with K I 's in the low picomolar range that compete with substrate binding in the S1 site. Alanine scanning of the loops surrounding the protease active site provide a rationale for inhibitor specificity. Each antibody binds to a number of residues flanking the active site, forming a unique three-dimensional binding epitope. Interestingly, one inhibitor binds in the active site cleft in a substrate-like manner, can be processed by MT-SP1 at low pH, and is a standard mechanism inhibitor of the protease. The mechanisms of inhibition provide a rationale for the effectiveness of these inhibitors, and suggest that the development of specific antibody-based inhibitors against individual members of closely related enzyme families is feasible, and an effective way to develop tools to tease apart complex biological processes.

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Microinjection of an antibody against the cysteine‐protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle

Cited by 13 publications

References 29 publications

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

Structure of an Fab–Protease Complex Reveals a Highly Specific Non-canonical Mechanism of Inhibition

The Mechanism of Inhibition of Antibody-based Inhibitors of Membrane-type Serine Protease 1 (MT-SP1)

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