Discípulos y maestros. Lo que aprendimos de Juan Rodríguez Soriano / Disciples and teachers. What we learn from Juan Rodríguez Soriano

Juan Rodríguez Soriano (1933-2010) fue un eminente nefrólogo pediátrico español, formador de varias generaciones de pediatras y nefrólogos pediátricos. Estaba considerado internacionalmente como uno de los pioneros de la nefrología pediátrica y, referente, en el de las tubolopatías. Su prestigio en esa especialidad fue unánimemente reconocido. Realizó sus estudios de Medicina en la Universidad de Barcelona. En 1959, obtuvo, una beca para ampliar su formación en el Hópital des Enfants Malades de Paris. En 1963 fue admitido en el Albert Einstein College of Medicine de Nueva York, en el que identificó la acidosis tubular proximal renal como una entidad fisopatológica independiente. En 1970, se hizo cargo del Departamento de Pediatría del Hospital Universitario de Cruces, cerca de Bilbao. Alklí, junto a Alfredo Vallo, siguió investigando y publicando sobre muchos temas pediátricos pero, especialmente sobre diversos aspectos de sus dos grandes temas de interés, las pruebas de función renal y las grandes temas de interés, las pruebas de función renal y la tubulopatías. A lo largo de 30 años, ambos desarrollaron las bases para la creación de una auténtica “escuela científica” nefrológica pediátrica (AU)

Juan Rodríguez Soriano (1933-2010) was an eminent Spanish nephrologist, forming several generations of paediatricians and paediatric nephrologist. He was internationally regarded as one of the pioneers of paediatric nephrology, and reference in tubolopathies. His reputation in that field was unanimously recognized. He studied medicine at the University of Barcelona. In 1959, he won a scholarship to further their training in the Hospital des Enfants Malades in Paris. In 1963 he was admitted to the Albert Einstein College of Medicine in New York where identified the proximal renal tubular acidosis as an independent physiopathological entity. In 1970, he took charge of the Department of Pediatrics, University Hospital of Cruces, Bilbao. There, nex to to Alfredo Vallo, continued its research and publications on many paediatric topics, but especially on various aspects of its two main topics of interest: renal function test and tubulopathies. Over 30 years, both developed the basis for creating a true “scientific school” of the paediatric nephrology (AU)

Structural determinants required for apical sorting of an intestinal brush-border membrane protein.

The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI(DeltaST) (stalk-free) and pro-SI(DeltaMA) (membrane anchor-free), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI(DeltaMA) does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI(DeltaST) does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI(DeltaMA). Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala(37)-Pro(48)) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala(49)-Pro(57)) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala(37)-Pro(48) stretch and requires the presence of the membrane anchoring domain.

Zebrafish (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production and requires a critical aspartate residue for its function in amyloidogenesis.

Alzheimer's disease (AD) is characterized by the invariable accumulation of senile plaques composed of amyloid beta-peptide (Abeta). Mutations in three genes are known to cause familial Alzheimer's disease (FAD). The mutations occur in the genes encoding the beta-amyloid precursor protein (betaAPP) and presenilin (PS1) and PS2 and cause the increased secretion of the pathologically relevant 42 amino acid Abeta42. We have now cloned the zebrafish (Danio rerio) PS1 homologue (zf-PS1) to study its function in amyloidogenesis and to prove the critical requirement of an unusual aspartate residue within the seventh putative transmembrane domain. In situ hybridization and reverse PCR reveal that zf-PS1 is maternally inherited and ubiquitously expressed during embryogenesis, suggesting an essential housekeeping function. zf-PS1 is proteolytically processed to produce a C-terminal fragment (CTF) of approximately 24 kDa similar to human PS proteins. Surprisingly, wt zf-PS1 promotes aberrant Abeta42 secretion like FAD associated human PS1 mutations. The unexpected pathologic activity of wt zf-PS1 may be due to several amino acid exchanges at positions where FAD-associated mutations have been observed. The amyloidogenic function of zf-PS1 depends on the conserved aspartate residue 374 within the seventh putative transmembrane domain. Mutagenizing this critical aspartate residue abolishes endoproteolysis of zf-PS1 and inhibits Abeta secretion in human cells. Inhibition of Abeta secretion is accompanied by the accumulation of C-terminal fragments of betaAPP, suggesting a defect in gamma-secretase activity. These data provide further evidence that PS proteins are directly involved in the proteolytic cleavage of betaAPP and demonstrate that this function is evolutionarily conserved.

One dose ceftriaxone vs. ten days of amoxicillin/clavulanate therapy for acute otitis media: clinical efficacy and change in nasopharyngeal flora.

OBJECTIVE: To compare the efficacy and the safety of a single intramuscular dose of ceftriaxone, 50 mg/kg, vs. a 10-day course of amoxicillin/clavulanate (amox/clav) therapy, 80 mg/kg/day of amoxicillin: 10 mg/kg/day of clavulanate in three divided doses, in children with acute otitis media (AOM) and to evaluate the changes in nasopharyngeal flora after treatment. METHODS: In a prospective, comparative, open randomized, multicenter trial, children were scheduled to return for visits on Days 12 to 14 (main end point) and Days 28 to 42 after the beginning of treatment for AOM. A nasopharyngeal swab for bacterial culture was obtained before the treatment and at Days 12 to 14. RESULTS: Between February, 1995, and May, 1996, 513 children with a mean age of 14.2 +/- 6.7 months were enrolled. All the patients were evaluable for the safety and intent-to-treat analyses and 463 for the per protocol efficacy. At Days 12 to 14 clinical success was obtained in 186 of the 235 children (79%) given ceftriaxone and in 188 of the 228 children (82.5%) treated with amox/clav. Among the patients with clinical success on Days 12 to 14, the success was maintained at Days 28 to 42 for 108 of 183 (59%) patients in the ceftriaxone group and 103 of 187 (55%) patients in the amox/clav group. Before the antibiotic treatment the percentages of children carrying Streptococcus pneumoniae (59.1%), Haemophilus influenzae (39.4%), Moraxella catarrhalis (55.7%) and the rate of penicillin-resistant S. pneumoniae (52.2%) were comparable between the 2 groups. At Days 12 to 14 the carriage of S. pneumoniae and M. catarrhalis was significantly different between the patients treated with ceftriaxone, 43.9 and 42.2, respectively, and the patients treated with amox/clav, 17.4 and 11.1%, respectively. Among the children carrying S. pneumoniae at Days 12 to 14, the percentage of penicillin-resistant strains reached 63.4% in the ceftriaxone treatment group and 83.0% in the amox/clav treatment group, (P = 0.02). Adverse events (mainly diarrhea) related to the study medication were reported more frequently (P < 0.0001) in the amox/clav treatment group. CONCLUSIONS: In an area with a high rate of penicillin-resistant S. pneumoniae, a single dose of ceftriaxone is as efficient as a 10-day course of amox/clav in the treatment of AOM in young children. There was for the two regimens an increased rate of penicillin-resistant strains among the pneumococci carried, whereas the chance for a child to carry a penicillin resistant S. pneumoniae did not increase after treatment.

Analysis of presenilin 1 and presenilin 2 expression and processing by newly developed monoclonal antibodies.

Because distinct mutations in presenilin 1 and presenilin 2 are a major cause of early-onset familial Alzheimer's disease, we generated four monoclonal antibodies for the identification, localization, and investigation of presenilins in various cell lines and tissues from patients and controls. We show that these antibodies are specific for the N- and C-terminal domains of human presenilin 1 and presenilin 2. They recognize presenilin full-length proteins and their approximately 28-35 kDa N-terminal fragments and approximately 18-20 kDa C-terminal fragments. None of the antibodies showed cross-reaction in their specific detection ability. We demonstrated that presenilin 1 and presenilin 2 are proteolytically processed in human glioma cell lines, transfected and untransfected human neuroblastoma SH-SY5Y cells, COS-7 cells, rat cerebellar neuronal ST15 cells, mouse and human brain. Remarkably, we observed that presenilin 2 is alternatively cleaved during apoptosis, producing smaller C-terminal fragments. By analyzing the subcellular distribution of presenilins, we found reticular and fine vesicular staining throughout the cell bodies. In addition, staining of Golgi compartments and the perinuclear envelope was observed. Alzheimer's disease brain showed strong immunoreactivity of presenilin 1 in reactive astrocytes and senile plaques. This high expression of presenilin 1 may explain the increased production and accumulation of the amyloid-beta peptide in patients with sporadic Alzheimer's disease in the absence of familial presenilin mutation.

Phosphorylation of presenilin-2 regulates its cleavage by caspases and retards progression of apoptosis.

Mutations within the Presenilin-2 (PS-2) gene are associated with early onset familial Alzheimer's disease. The gene encodes a polytopic transmembrane protein that undergoes endoproteolytic processing resulting in the generation of N-terminal and C-terminal fragments (CTFs). PS-2 is also cleaved by proteases of the caspase family during apoptotic cell death. CTFs of PS-2 were shown to inhibit apoptosis, suggesting an important role in the regulation of programmed cell death. Recently, we found that the CTF of PS-2 is phosphorylated in vivo. We mapped the in vivo phosphorylation sites of PS-2 to serine residues 327 and 330, which are localized immediately adjacent to the cleavage sites of caspases after aspartate residues 326 and 329. Phosphorylation of PS-2 inhibits its cleavage by caspase-3. This effect can be mimicked by substitutions of serines 327 and 330 by aspartate or glutamate. In addition, the uncleavable form of PS-2 CTF was found to enhance its antiapoptotic properties, leading to a slower progression of apoptosis. These results demonstrate that PS-2 cleavage as well as its function in apoptosis can be regulated by protein phosphorylation. Alterations in the phosphorylation of PS-2 may therefore promote the pathogenesis of AD by affecting the susceptibility of neurons to apoptotic stimuli.

Truncated presenilin 2 derived from differentially spliced mRNA does not affect the ratio of amyloid beta-peptide 1-42/1-40.

Numerous mutations in the presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD). Here we characterize the expression of two naturally occurring alternative PS2 transcripts which lack either exons 3 and 4 (PS2 deltaexon3,4) or exons 3, 4, and 8 (PS2 deltaexon3,4,8). These transcripts do not contain the natural initiation codon within exon 3. The transcripts are efficiently translated as N-terminal truncated proteins. These deleted proteins are still able to regulate formation of endogenous PS fragments, indicating that the C-terminal half of the PS2 protein is sufficient for this phenomenon. Although approximately 50% of the PS1 and both PS2 mutations occur within the N-terminal region lacking in the PS2 deltaexon3,4 and PS2 deltaexon3,4,8 proteins, expression of these truncated proteins does not affect pathological generation of amyloid beta-peptide (Abeta). This suggests that point mutations causing AD are gain of function mutations.

Proteolytic processing of Alzheimer's disease associated proteins.

Amyloid beta-peptide (A beta), the major component of senile plaques, is generated by proteolytic processing from the beta-amyloid precursor protein (beta APP). Mutations within the beta APP gene cause early onset familial AD (FAD) by affecting A beta generation. Interestingly, the much more abundant mutations within the presenilin (PS) genes also result in the abnormal generation of a 42 residue A beta (A beta 42), thus clearly supporting a pivotal role of A beta for the pathology of AD. PS proteins are proteolytically processed into stable 30 kDa N-terminal fragments (NTF) and 20 kDa C-terminal fragments (CTF). Beside the conventional proteolytic pathway. PS proteins can also be cleaved further C-terminal by proteases of the caspase superfamily. PS proteins were localized within the endoplasmic reticulum (ER) and early Golgi, compartments which we have demonstrated to be involved in A beta 42 generation and intracellular accumulation. Using Caenorhabditis elegans as a simple animal model, we demonstrate that PS proteins are involved in NOTCH signaling FAD causing mutations interfere with the biological function of PS proteins in NOTCH signaling.

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

Caspase-mediated cleavage is not required for the activity of presenilins in amyloidogenesis and NOTCH signaling.

The Alzheimer's disease (AD) associated presenilin (PS) proteins are proteolytically processed. One of the processing pathways involves cleavage by caspases. Pharmacological inhibition of caspases is currently being discussed as a treatment for a variety of neurodegenerative diseases, including AD. We therefore inhibited caspase mediated processing of PS-1 and PS-2 in cells transfected with wt and mutant PS by mutagenizing the substrate recognition site or by using specific peptide aldehydes known to block caspases. We found that the inhibition of caspase mediated processing of PS proteins does not decrease its amyloidogenic activity. PS cDNA constructs with mutations in the caspase cleavage site are biologically active in Caenorhabditis elegans such as the wt human PS proteins, demonstrating that caspase-mediated cleavage is not required for the physiological PS function in NOTCH signaling.

Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms.

The majority of familial Alzheimer's disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N-terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its approximately 20 kDa CTF involving protein kinase C (PKC) and/or protein kinase A (PKA). We now have found that the CTF of the highly homologous PS-2 is also phosphorylated. Surprisingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, the PS-2 CTF is constitutively phosphorylated in vivo by serine/threonine protein kinases, which are independent of phorbol ester and intracellular cAMP. In vitro the large hydrophilic loop of PS-2 between transmembrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK-1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed that the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosphorylate the CTF of PS-2 within its hydrophilic loop domain in vivo. Interestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.

Alzheimer's disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease leading to the formation of two stable fragments of approximately 30 and approximately 20 kDa [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. In addition to the conventional fragments, alternative cleavage products were observed as well. Here we characterize an alternative proteolytic pathway of PS-1 which involves proteases of the caspase superfamily. Caspase mediated cleavage occurs between aspartate 345 and serine 346 C-terminal to the conventional cleavage determined previously [Podlisny, M., et al., (1997) Neurobiol. Dis. 3, 325-337]. Full-length PS-1 can serve as a substrate for caspase-like proteases, as demonstrated by the generation of the alternative C-terminal fragment in cells expressing PS-1 containing the Deltaexon 10 deletion which is known to accumulate as a full-length molecule [Thinakaran, G., et al. (1996)]. By inhibition of de novo protein synthesis in untransfected cells we demonstrate that the conventional C-terminal fragment of PS-1 is a substrate for caspase-like proteases as well. Therefore full-length and the conventional C-terminal fragment of PS-1 can serve as potential death substrates. Due to the fact that very little full-length PS-1 is expressed in vivo, the much more abundant C-terminal fragment and not the full-length precursor is the major in vivo substrate for the alternative cleavage of PS-1 by proteases of the caspase superfamily.

The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756-760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins.

Pediatric nephrology workforce in Latin America.

Pediatric nephrology workforce issues were examined in a Latin American survey involving 14 countries. The number of children under 15 years per pediatric nephrologist varied widely among countries: Argentina, Cuba, Venezuela, and Uruguay had an unusually high number of pediatric nephrologists. Guatemala represents the opposite end of the spectrum of values (1,582.6 thousand children under 15 years per pediatric nephrologist). A significant inverse correlation was found between children under 15 years per pediatric nephrologist and national gross domestic product per capita (r=-0.52, P<0.05) and a significant correlation between children per pediatric nephrologist and infant mortality (r=0.82, P<0.005, Spearman's rank correlation coefficient). The same correlations were observed for total population per pediatric nephrologist. However, the pediatric nephrology workforce does not merely reflect national economic status. Official health care policies, market forces, and social regulations also have an influence. A study of the number of pediatric nephrologists necessary for adequate planning of care of children with renal disease in Latin America is urgently needed.

Cellular expression and proteolytic processing of presenilin proteins is developmentally regulated during neuronal differentiation.

We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected approximately 34-kDa N-terminal proteolytic fragment of presenilin-1 and the approximately 38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a approximately 38-kDa fragment for presenilin-1 and a approximately 42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.

Presenilins are processed by caspase-type proteases.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.

Polarised expression of human intestinal N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin) alpha and beta subunits in Madin-Darby canine kidney cells.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C.

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

Human presenilin-1, but not familial Alzheimer's disease (FAD) mutants, facilitate Caenorhabditis elegans Notch signalling independently of proteolytic processing.

The majority of cases with familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes show considerable sequence similarity to the sel-12 gene of Caenorhabditis elegans, which has been postulated to function in the facilitated signalling by lin-12 and glp-1. In order to analyse the functional conservation of the presenilins, we introduced the human PS-1 cDNA, as well as clinical and deletion mutant proteins, into sel-12 mutant animals and tested their potential to rescue the egg-laying defect. Human PS-1 expressed from the sel-12 promoter fully rescued the sel-12 phenotype, whereas two missense mutations, C410Y and A246E, identified in pedigrees with FAD, exhibited a strongly decreased rescuing activity. The large hydrophilic loop and transmembrane domain 7 are required for the biological activity of PS-1. PS-1 protein was proteolytically cleaved in C. elegans as it is in human cells. A PS-1 splice variant (FAD mutation deltaexon9) that does not undergo proteolytic cleavage also substituted for sel-12. The conservation of function of human PS-1 and C. elegans sel-12 suggests that presenilin proteins are required, directly or indirectly, for the proper operation of the Notch signalling pathway. FAD-associated mutant proteins tested showed different rescuing activities, indicating that they might affect different functional or regulatory aspects of PS-1. Proteolytic processing is not a prerequisite for PS-1 function in C. elegans.

The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid beta protein ending at the 42nd (or 43rd) residue.

To gain insights into the significance of presenilins (PS) in the pathogenetic mechanisms of early-onset familial Alzheimer disease (FAD), we expressed cDNAs for wild-type PS2 and PS2 with the Volga German (N141I) mutation in cultured cells and then examined the metabolism of the transfected proteins and their effect on the C-terminal properties of secreted amyloid beta protein (A beta). PS2 was identified as a 50- to 55-kDa protein, which was cleaved to produce N-terminal fragments of 35-40 kDa and C-terminal fragments of 19-23 kDa. The Volga German (N141I) mutation did not cause any significant change in the metabolism of PS2. COS-1 cells doubly transfected with cDNAs for N141I mutant PS2 and human beta-amyloid precursor protein (betaAPP) or a C-terminal fragment thereof, as well as mouse Neuro2a neuroblastoma cells stably transfected with N141I mutant PS2 alone, secreted 1.5- to 10-fold more A beta ending at residues 42 (or 43) [A beta42(43)] compared with those expressing the wild-type PS2. These results strongly suggest that the PS2 mutation (N141I) linked to FAD alters the metabolism of A beta/betaAPP to foster the production of the form of A beta that most readily deposits in amyloid plaques. Thus, mutant PS2 may lead to AD by altering the metabolism of A beta/betaAPP.