RESUMO
The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.
Assuntos
Secretases da Proteína Precursora do Amiloide , Animais , Feminino , Masculino , Camundongos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Presenilina-1/genética , Estrutura Terciária de ProteínaRESUMO
Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.
Assuntos
Secretases da Proteína Precursora do Amiloide , Complexos Multiproteicos , Presenilina-1 , Sulfonamidas , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/antagonistas & inibidores , Presenilina-1/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Sulfonamidas/farmacologia , Especificidade por Substrato , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismoRESUMO
Given the high frequency of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL), inhibition of the γ-secretase complex remains an attractive target to prevent ligand-independent release of the cytoplasmic tail and oncogenic NOTCH1 signaling. However, four different γ-secretase complexes exist, and available inhibitors block all complexes equally. As a result, these cause severe "on-target" gastrointestinal tract, skin, and thymus toxicity, limiting their therapeutic application. Here, we demonstrate that genetic deletion or pharmacologic inhibition of the presenilin-1 (PSEN1) subclass of γ-secretase complexes is highly effective in decreasing leukemia while avoiding dose-limiting toxicities. Clinically, T-ALL samples were found to selectively express only PSEN1-containing γ-secretase complexes. The conditional knockout of Psen1 in developing T cells attenuated the development of a mutant NOTCH1-driven leukemia in mice in vivo but did not abrogate normal T cell development. Treatment of T-ALL cell lines with the selective PSEN1 inhibitor MRK-560 effectively decreased mutant NOTCH1 processing and led to cell cycle arrest. These observations were extended to T-ALL patient-derived xenografts in vivo, demonstrating that MRK-560 treatment decreases leukemia burden and increased overall survival without any associated gut toxicity. Therefore, PSEN1-selective compounds provide a potential therapeutic strategy for safe and effective targeting of T-ALL and possibly also for other diseases in which NOTCH signaling plays a role.
Assuntos
Terapia de Alvo Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Receptores Notch/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Trato Gastrointestinal/patologia , Deleção de Genes , Marcação de Genes , Humanos , Masculino , Camundongos , Presenilina-1/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
Amyloids are often associated with pathologic processes such as in Alzheimer's disease (AD), but can also underlie physiological processes such as pigmentation. Formation of pathological and functional amyloidogenic substrates can require precursor processing by proteases, as exemplified by the generation of Aß peptide from amyloid precursor protein (APP) by beta-site APP cleaving enzyme (BACE)1 and γ-secretase. Proteolytic processing of the pigment cell-specific Melanocyte Protein (PMEL) is also required to form functional amyloid fibrils during melanogenesis, but the enzymes involved are incompletely characterized. Here we show that the BACE1 homologue BACE2 processes PMEL to generate functional amyloids. BACE2 is highly expressed in pigment cells and Bace2(-/-) but not Bace1(-/-) mice display coat color defects, implying a specific role for BACE2 during melanogenesis. By using biochemical and morphological analyses, combined with RNA silencing, pharmacologic inhibition, and BACE2 overexpression in a human melanocytic cell line, we show that BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain, releasing the PMEL luminal domain into endosomal precursors for the formation of amyloid fibrils and downstream melanosome morphogenesis. These studies identify an amyloidogenic substrate of BACE2, reveal an important physiological role for BACE2 in pigmentation, and highlight analogies in the generation of PMEL-derived functional amyloids and APP-derived pathological amyloids.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloide/biossíntese , Ácido Aspártico Endopeptidases/metabolismo , Melanossomas/metabolismo , Antígeno gp100 de Melanoma/metabolismo , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Células HeLa , Humanos , Melaninas/biossíntese , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epitélio Pigmentado Ocular/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Pigmentação da Pele/genética , Pigmentação da Pele/fisiologiaRESUMO
Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ß (Aß) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aß generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aß production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Presenilinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid ß (Aß)42 relative to Aß40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aß generation via three different mechanisms, resulting in qualitative changes in the Aß profiles, which are not limited to Aß42. Loss of É-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial É-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aß products, and suggest fundamental improvements for current drug development efforts.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloide/metabolismo , Presenilina-1/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Receptores ErbB/metabolismo , Cinética , Receptor ErbB-4 , Receptor Notch1/metabolismoRESUMO
The gamma-secretase complex plays a role in Alzheimer's disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the gamma-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different gamma-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B gamma-secretase in a mouse Alzheimer's disease model led to improvements of Alzheimer's disease-relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total gamma-secretase activity in the human brain, and thus specific targeting of Aph1B-containing gamma-secretase complexes may help generate less toxic therapies for Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Endopeptidases/metabolismo , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Endopeptidases/química , Endopeptidases/genética , Feminino , Humanos , Aprendizagem em Labirinto , Proteínas de Membrana/metabolismo , Memória , Camundongos , Neurônios/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptor Notch1/metabolismo , Transdução de SinaisRESUMO
Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.
Assuntos
Apoptose/fisiologia , Citocromos c/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Caquexia/genética , Linhagem Celular , Células Cultivadas , Regulação para Baixo/genética , Feminino , GTP Fosfo-Hidrolases/genética , Genes Letais , Linfócitos/metabolismo , Linfócitos/patologia , Metaloproteases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismoRESUMO
A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.
Assuntos
Cóclea/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Externas/ultraestrutura , Receptores de Estrogênio/deficiência , Animais , Western Blotting , Modelos Animais de Doenças , Genótipo , Perda Auditiva/congênito , Perda Auditiva/patologia , Ácido Hialurônico/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Uridina Difosfato Glucose Desidrogenase/metabolismoRESUMO
We investigated whether peroxisome proliferator-activated receptor gamma (PPARgamma) could be involved in the modulation of the amyloid cascade causing Alzheimer's disease. Inducing expression or activating PPARgamma using synthetic agonists of the thiazolinedione family results in a dramatic decrease in the levels of the amyloid-beta (Abeta) peptide in the conditioned medium of neuronal and non-neuronal cells. PPARgamma does not affect expression or activity of any of the secretases involved in the generation of the Abeta peptide but induces a fast, cell-bound clearing mechanism responsible for the removal of the Abeta peptide from the medium. Although PPARgamma expression is generally low in the CNS, induction of PPARgamma expression during inflammation could be beneficial for inducing Abeta clearance. We confirm that the Abeta clearance mechanism can indeed be induced by PPARgamma activation in primary murine-mixed glia and cortical neuronal cultures. Our results suggest that PPARgamma-controlled mechanisms should be explored further as potential drug targets for Alzheimer's disease treatment.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Cromanos/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , PPAR gama/fisiologia , Tiazolidinedionas/farmacologia , Secretases da Proteína Precursora do Amiloide , Anilidas/farmacologia , Animais , Ácido Aspártico Endopeptidases , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Meios de Cultivo Condicionados/química , Endopeptidases/análise , Endopeptidases/metabolismo , Humanos , Rim , Camundongos , Neuroblastoma/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , PPAR gama/genética , Fragmentos de Peptídeos/metabolismo , Pioglitazona , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptor Notch1 , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/fisiologia , Receptores X de Retinoides/efeitos dos fármacos , Rosiglitazona , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , TroglitazonaRESUMO
Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.
Assuntos
Autofagia/fisiologia , Hipocampo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Actinas/genética , Actinas/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases , Proteína 12 Relacionada à Autofagia , Catepsina D/genética , Moléculas de Adesão Celular , Endopeptidases/genética , Endopeptidases/metabolismo , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Hipocampo/ultraestrutura , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Presenilina-1 , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
Presenilin (PS1) and (PS2) are the centers of gamma-secretase that release Abeta from APP in Alzheimer's disease (AD). They cleave signaling proteins like Notch and downregulate beta-catenin to modulate Wnt signaling. Inactivation of PS1 or PS1 and PS2 causes a prenatally lethal 'Notch phenotype,' which has hampered investigation of PS function in adulthood seriously. We have thus turned towards PS1+/-PS2-/- mice which carry the most severe reduction of PS alleles compatible with survival, to analyze the consequences of impaired PS function especially in adulthood. In these 'partial deficient' mice, PS1 protein concentration is considerably lowered, functionally reflected by reduced gamma-secretase activity and impaired beta-catenin downregulation. Their phenotype is normal up to approximately 6 months, when the majority of the mice develop an autoimmune disease characterized by dermatitis, glomerulonephritis, keratitis and vasculitis, as seen in human systemic lupus erythematosus. Besides B-cell dominated infiltrates, we observe a hypergammaglobulinemia with immune complex deposits in several tissues, high-titer nuclear autoantibodies and an increased CD4+/CD8+ ratio. The mice further develop a benign skin hyperplasia similar to human seborrheic keratosis as opposed to malignant keratocarcinomata observed in skin-specific PS1 'full' knockouts. A partial reduction of PS function in PS1+/-PS2-/- mice causes a novel phenotype in adulthood unrelated to the developmental defects of full knockouts. As PS1+/-PS2+/- mice remain healthy, this points towards a sharply defined minimum of PS function. Skin and immune system appear to be especially sensitive targets of impaired PS function and may need careful monitoring if gamma-secretase inhibitors are envisaged for treating AD.
Assuntos
Doenças Autoimunes/genética , Ceratose Seborreica/genética , Proteínas de Membrana/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Animais , Anticorpos Antinucleares/imunologia , Ácido Aspártico Endopeptidases , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proteínas de Transporte/metabolismo , Endopeptidases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ceratose Seborreica/imunologia , Ceratose Seborreica/metabolismo , Ceratose Seborreica/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Presenilina-1 , Presenilina-2 , Nexinas de Proteases , Receptores de Superfície Celular , Receptores Notch , Proteínas WntRESUMO
Presenilin (PS) is essential for gamma-cleavage, which is required for the generation of amyloid beta-protein (Abeta) from the beta-amyloid precursor protein. However, it remains to be clarified how gamma-cleavage is regulated. To elucidate the regulation of PS-mediated gamma-cleavage, we developed a new functional screening method for identifying cDNA that enhances gamma-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances gamma-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and Abeta assay screening. With this screening method, we isolated several cDNAs enhancing gamma-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of Abeta generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in gamma-cleavage in the Abeta generation pathway, including modulators of gamma-secretase activity or the intracellular trafficking of factors necessary for gamma-cleavage.