Vicente Rubio - Academia.edu (original) (raw)

Papers by Vicente Rubio

Research paper thumbnail of Detection of SARS-CoV-2 in a dog with hemorrhagic diarrhea

BMC Veterinary Research

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of C... more Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequen...

Research paper thumbnail of Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic

Frontiers in Microbiology

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-C... more After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and ...

Research paper thumbnail of Suggested guidelines for the diagnosis and management of urea cycle disorders

Orphanet Journal of Rare Diseases, 2012

Research paper thumbnail of Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly. Corrigendum

IUCrJ

The article by Casino et al. [IUCrJ (2018). 5, 765–779] is corrected.

Research paper thumbnail of The Conserved Family of the Pyridoxal Phosphate-Binding Protein (PLPBP) and Its Cyanobacterial Paradigm PipY

Life

The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservati... more The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservation and is represented in all three domains of life. PLPBP members, of which a few representatives have been studied in different contexts, are single-domain proteins with no known enzymatic activity that exhibit the fold type III of PLP-holoenzymes, consisting in an α/β barrel (TIM-barrel), where the PLP cofactor is solvent-exposed. Despite the constant presence of cofactor PLP (a key catalytic element in PLP enzymes), PLPBP family members appear to have purely regulatory functions affecting the homeostasis of vitamin B6 vitamers and amino/keto acids. Perturbation of these metabolites and pleiotropic phenotypes have been reported in bacteria and zebrafish after PLPBP gene inactivation as well as in patients with vitamin B6-dependent epilepsy that results from loss-of-function mutations at the PLPBP. Here, we review information gathered from diverse studies and biological systems, emphasi...

Research paper thumbnail of Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures

Journal of Inherited Metabolic Disease, 2021

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (P... more Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five Alvaro Briso-Montiano and Francisco del Caño-Ochoa contributed equally to the work.

Research paper thumbnail of Δ1‐Pyrroline‐5‐carboxylate synthetase deficiency: An emergent multifaceted urea cycle‐related disorder

Journal of Inherited Metabolic Disease, 2020

The bifunctional homooligomeric enzyme Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and its encod... more The bifunctional homooligomeric enzyme Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa, and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015 to 2016, an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity, or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations wi...

Research paper thumbnail of Structural and Functional Insights into Endoglin Ligand Recognition and Binding

PLoS ONE, 2012

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascu... more Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-b receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-b signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Research paper thumbnail of Estimation of the total number of disease‐causing mutations in ornithine transcarbamylase (OTC) deficiency. Value of the OTC structure in predicting a mutation pathogenic potential

Journal of Inherited Metabolic Disease, 2007

Ornithine transcarbamylase deficiency (OTCD), the X‐linked, most frequent urea cycle error, resul... more Ornithine transcarbamylase deficiency (OTCD), the X‐linked, most frequent urea cycle error, results from mutations in the OTC gene, encoding a 354‐residue polypeptide. To date 341 OTCD clinical mutations, including 222 missense single nucleotide changes (mSNCs), have been compiled (Hum Mutat 2006;27:626). OTCD mutation detection might be simplified if the entire repertoire of OTCD‐causing mutations were known. We estimate the size of this repertoire from 23 new OTCD patients exhibiting 22 different mutations, of which 9, including 4 mSNCs, are novel. The complete repertoire of OTCD‐causing mutations is estimated as 560 mutations (95% confidence interval, 422–833 mutations), including 290 mSNCs (95% confidence interval, 230–394 mSNCs). Thus, OTCD diagnosis based on the screening for known mutations might attain ˜90% sensitivity in <5 years. Since disease‐causing mSNCs represent <20% of the 2064 possible OTC mSNCs, simple approaches are essential for discrimination between causa...

Research paper thumbnail of Mutant alleles associated with late-onset ornithine transcarbamylase deficiency in male patients have recurrently arisen and have been retained in some populations

Journal of Human Genetics, 2009

We performed haplotype analysis using nine single nucleotide polymorphisms in the ornithine trans... more We performed haplotype analysis using nine single nucleotide polymorphisms in the ornithine transcarbamylase gene to explore the ancestral origins of three mutations associated with late-onset phenotype in male patients: p.R40H, p.R277W and p.Y55D. Overall, 8 haplotypes were defined among 14 families carrying p.R40H, 5 families carrying p.R277W and 2 families with p.Y55D mutations. Of nine Japanese families carrying p.R40H, eight exhibited haplotype (HT)1, whereas the other family harbored HT2. Among three Caucasian families, one Spanish and one Australian family bore HT3; one Austrian family had HT4. Two US patients harbored HT2 and HT4. Among families carrying p.R277W, HT5 was found in one Japanese, one Korean and one US family. Two other US families had HT2 and HT6. Two families carrying p.Y55D, both Japanese, shared HT1. These results indicate that the p.R40H mutation has arisen recurrently in all populations studied, although there is evidence for a founder effect in Japan, with most cases probably sharing a common origin, and to a lesser extent in subjects of European ancestry (HT3). It is evident that p.R277W mutation has recurred in discrete populations. The p.Y55D mutation appears to have arisen from a common ancestor, because this transversion (c.163T4G) occurs rarely.

Research paper thumbnail of Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures

Journal of Histochemistry & Cytochemistry, 1990

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a ke... more Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.

Research paper thumbnail of Activation of carbamoyl phosphate synthetase from Escherichia coli by glycerol

Biochemical and Biophysical Research Communications, 1982

Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryopro... more Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryoprotectants. Their diverse chemical structure and the normal stoichiometry andpattern of positional isotopic exchange for the reaction catalyzed in their presence strongly supports an allosteric mechanism for activation by the physiological activator, acetylglutamate, and by the cryoprotectants. Activation appears not to require any specific group in the cryoprotectants, but different agents activate to different degrees. All cryoprotectants found to activate are good hydrogen-bond formers and have low molecular weight (<1 000 for polyethylene glycols). This suggests that the cryoprotectant must associate with the enzyme to activate. In the presence of acetylglutamate all cryoprotectants tested, irrespective of their capacity to activate, inhibit. Carbamoyl phosphate synthetase I has activity in the absence of cryoprotectants and acetylglutamate. Under these conditions the Km's for ATP and K + are very high, the K m for NH + is very low, the Vma x appears not to exceed 15% of that at saturation of acetylglutamate and the ratio of ATPase to carbamoyl phosphate synthetase activity is ca. three times that of the acetylglutamate activated enzyme. Acetylglutamate lowers the Km's for ATP and K +, and increases the K m for NH + and the Vma x. From steady state kinetics, pulse-chase, and measurements of the rate of activation, an allosteric model for activation of the enzyme by acetylglutamate is proposed in which there are two active species, R and R • AG (AG = acetylglutamate), which have similar but not identical activities. One molecule of ATP binds with high affinity to active and inactive enzyme species, but K + and the other molecule of ATP bind preferentially to the active species. In the absence of acetylglutamate, glycerol (an effective activator) reduces the Km's for ATP and K +, increases the Vma x, and dramatically reduces the S0. 5 for acetylglutamate to <1 #M for the synthetase reaction. In the presence of excess acetylglutamate, glycerol markedly reduces the K m for ATP for the synthetase, decreases the Vmax, NH + becomes a more effective activator than K +, and Na + activates appreciably. From these and other kinetic measurements on the ATPase activity, the partial reverse reaction and rates of activation, and from pulse chase observations, the effect of glycerol is rationalized in terms of the allosteric model proposed for the actions of acetylglutamate if: a) glycerol reduces the rate of transformation and alters the allosteric equilibria between active and inactive enzyme species, and b) glycerol inhibits the active enzyme, at least in part due to slower conformational changes associated with dissociation of the products and binding of substrates. Glycerol also activates carbamoyl phosphate synthetase from E. coli in the absence of ornithine. This and the above findings are compared with data on the actions of cryoprotectants on carbamoyl phosphate synthetase II (EC 6.3.5.5) from rat liver. There are considerable similarities in the actions of these agents on the three enzymes. The relationship between these findings and the actions of cryoprotectants on other enzymes and possible physiological implications are discussed.

Research paper thumbnail of P5CS expression study in a new family with ALDH18A1‐associated hereditary spastic paraplegia SPG9

Annals of Clinical and Translational Neurology, 2019

In 2015–2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SP... more In 2015–2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease‐causing mechanisms. We now describe a baculovirus–insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease‐causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits.

Research paper thumbnail of Insight into vitamin B6-dependent epilepsy due toPLPBP(previouslyPROSC) missense mutations

Human Mutation, 2018

Vitamin B 6-dependent genetic epilepsy was recently associated to mutations in PLPBP (previously ... more Vitamin B 6-dependent genetic epilepsy was recently associated to mutations in PLPBP (previously PROSC), the human version of the widespread COG0325 gene that encodes TIM-barrel-like pyridoxal phosphate (PLP)-containing proteins of unclear function. We produced recombinantly, purified and characterized human PROSC (called now PLPHP) and its six missense mutants reported in epileptic patients. Normal PLPHP is largely a monomer with PLP bound through a Schiff-base linkage. The PLP-targeting antibiotic Dcycloserine decreased the PLP-bound peak as expected for pseudo-first-order reaction. The p.Leu175Pro mutation grossly misfolded PLPHP. Mutations p.Arg241Gln and p.Pro87Leu decreased protein solubility and yield of pure PLPHP, but their pure forms were well folded, similarly to pure p.Pro40Leu, p.Tyr69Cys and p.Arg205Gln mutants (judged from CD spectra). PLPHP stability was decreased in p.Arg241Gln, p.Pro40Leu and p.Arg205Gln mutants (thermofluor assays). The p.Arg241Gln and p.Tyr69Cys mutants respectively lacked PLP or had a decreased amount of this cofactor. With p.Tyr69Cys there was extensive protein dimerization due to disulfide bridge formation, and PLP accessibility was decreased (judged from D-cycloserine reaction). A 3-D model of human PLPHP allowed rationalizing the effects of most mutations. Overall, the six missense mutations caused ill effects and five of them impaired folding or decreased stability, suggesting the potential of pharmacochaperonebased therapeutic approaches.

Research paper thumbnail of PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

Frontiers in microbiology, 2017

Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyan... more Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essentia...

Research paper thumbnail of Happy microbes in hostile niches. A symposium on extremophiles

International Microbiology, Mar 1, 2004

Happy microbes in hostile niches. A symposium on extremophiles Belgium can boast the privilege of... more Happy microbes in hostile niches. A symposium on extremophiles Belgium can boast the privilege of having had a microbiologist Nobel laureate (Physiology or Medicine, 1919), Jules-Jean-Baptiste-Vincent Bordet. Today, the country's outstanding microbiological tradition remains alive and well at the Research Institute of the CERIA/COOVI (1 Av. Emile Gryzon, Brussels), the center where Nicolas Glansdorff has worked for most of his scientific career and of which he was director (Fig. 1). The French-speaking-community moiety of this institute is now named Institut de Recherche Jean Marie Wiame, to honor its founding father, who was also the scientific mentor of Nicolas Glansdorff. Werner Maas (New York University) and Luigi Gorini (Harvard University) were Glansdorff's mentors in the United States. Happy Microbes, convened to honor the officially retiring Nicolas Glansdorff, paid tribute to his paramount interests in both extremophiles and the origin of life. The meeting was held on September 27, 2003, under the auspices of the Belgian Society for Microbiology, in the Etterbeek Campus

Research paper thumbnail of ALDH18A1gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism

Brain, 2015

Sir, We have read with great interest the article published recently in Brain by Coutelier et al.... more Sir, We have read with great interest the article published recently in Brain by Coutelier et al. (2015) 'Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia'. In support of the message of this paper, we would like to report that mutations in ALDH18A1, the causative gene in Coutelier's paper, are the cause of SPG9 (MIM#601162), a rare form of autosomal dominant hereditary spastic paraplegia (HSP) complicated with vomiting and congenital bilateral cataracts

Research paper thumbnail of SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules

Surface plasmon resonance monitoring of the binding of transcription factors CRP and NtcA from Sy... more Surface plasmon resonance monitoring of the binding of transcription factors CRP and NtcA from Synechocystis sp. PCC6803 to promoter fragments of glnA, glnN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity

Research paper thumbnail of Suggested guidelines for the diagnosis and management of urea cycle disorders

Orphanet Journal of Rare Diseases, 2012

Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to... more Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to defects affecting the catalysts of the Krebs-Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial ornithine/citrulline antiporter) with an estimated incidence of 1:8.000. Patients present with hyperammonemia either shortly after birth (50%) or, later at any age, leading to death or to severe neurological handicap in many survivors. Despite the existence of effective therapy with alternative pathway therapy and liver transplantation, outcomes remain poor. This may be related to underrecognition and delayed diagnosis due to the nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity. These guidelines aim at providing a trans-European consensus to: guide practitioners, set standards of care and help awareness campaigns. To achieve these goals, the guidelines were developed using a Delphi methodology, by having professionals on UCDs across seven European countries to gather all the existing evidence, score it according to the SIGN evidence level system and draw a series of statements supported by an associated level of evidence. The guidelines were revised by external specialist consultants, unrelated authorities in the field of UCDs and practicing pediatricians in training. Although the evidence degree did hardly ever exceed level C (evidence from non-analytical studies like case reports and series), it was sufficient to guide practice on both acute and chronic presentations, address diagnosis, management, monitoring, outcomes, and psychosocial and ethical issues. Also, it identified knowledge voids that must be filled by future research. We believe these guidelines will help to: harmonise practice, set common standards and spread good practices with a positive impact on the outcomes of UCD patients.

Research paper thumbnail of Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine

Protein Science, 2008

The enzymes carbamoyl phosphate synthetase~CPS! and carbamate kinase~CK! make carbamoyl phosphate... more The enzymes carbamoyl phosphate synthetase~CPS! and carbamate kinase~CK! make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open b-sheet surrounded by a-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.

Research paper thumbnail of Detection of SARS-CoV-2 in a dog with hemorrhagic diarrhea

BMC Veterinary Research

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of C... more Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequen...

Research paper thumbnail of Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic

Frontiers in Microbiology

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-C... more After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and ...

Research paper thumbnail of Suggested guidelines for the diagnosis and management of urea cycle disorders

Orphanet Journal of Rare Diseases, 2012

Research paper thumbnail of Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly. Corrigendum

IUCrJ

The article by Casino et al. [IUCrJ (2018). 5, 765–779] is corrected.

Research paper thumbnail of The Conserved Family of the Pyridoxal Phosphate-Binding Protein (PLPBP) and Its Cyanobacterial Paradigm PipY

Life

The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservati... more The PLPBP family of pyridoxal phosphate-binding proteins has a high degree of sequence conservation and is represented in all three domains of life. PLPBP members, of which a few representatives have been studied in different contexts, are single-domain proteins with no known enzymatic activity that exhibit the fold type III of PLP-holoenzymes, consisting in an α/β barrel (TIM-barrel), where the PLP cofactor is solvent-exposed. Despite the constant presence of cofactor PLP (a key catalytic element in PLP enzymes), PLPBP family members appear to have purely regulatory functions affecting the homeostasis of vitamin B6 vitamers and amino/keto acids. Perturbation of these metabolites and pleiotropic phenotypes have been reported in bacteria and zebrafish after PLPBP gene inactivation as well as in patients with vitamin B6-dependent epilepsy that results from loss-of-function mutations at the PLPBP. Here, we review information gathered from diverse studies and biological systems, emphasi...

Research paper thumbnail of Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures

Journal of Inherited Metabolic Disease, 2021

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (P... more Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five Alvaro Briso-Montiano and Francisco del Caño-Ochoa contributed equally to the work.

Research paper thumbnail of Δ1‐Pyrroline‐5‐carboxylate synthetase deficiency: An emergent multifaceted urea cycle‐related disorder

Journal of Inherited Metabolic Disease, 2020

The bifunctional homooligomeric enzyme Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and its encod... more The bifunctional homooligomeric enzyme Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa, and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015 to 2016, an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity, or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations wi...

Research paper thumbnail of Structural and Functional Insights into Endoglin Ligand Recognition and Binding

PLoS ONE, 2012

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascu... more Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-b receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-b signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Research paper thumbnail of Estimation of the total number of disease‐causing mutations in ornithine transcarbamylase (OTC) deficiency. Value of the OTC structure in predicting a mutation pathogenic potential

Journal of Inherited Metabolic Disease, 2007

Ornithine transcarbamylase deficiency (OTCD), the X‐linked, most frequent urea cycle error, resul... more Ornithine transcarbamylase deficiency (OTCD), the X‐linked, most frequent urea cycle error, results from mutations in the OTC gene, encoding a 354‐residue polypeptide. To date 341 OTCD clinical mutations, including 222 missense single nucleotide changes (mSNCs), have been compiled (Hum Mutat 2006;27:626). OTCD mutation detection might be simplified if the entire repertoire of OTCD‐causing mutations were known. We estimate the size of this repertoire from 23 new OTCD patients exhibiting 22 different mutations, of which 9, including 4 mSNCs, are novel. The complete repertoire of OTCD‐causing mutations is estimated as 560 mutations (95% confidence interval, 422–833 mutations), including 290 mSNCs (95% confidence interval, 230–394 mSNCs). Thus, OTCD diagnosis based on the screening for known mutations might attain ˜90% sensitivity in <5 years. Since disease‐causing mSNCs represent <20% of the 2064 possible OTC mSNCs, simple approaches are essential for discrimination between causa...

Research paper thumbnail of Mutant alleles associated with late-onset ornithine transcarbamylase deficiency in male patients have recurrently arisen and have been retained in some populations

Journal of Human Genetics, 2009

We performed haplotype analysis using nine single nucleotide polymorphisms in the ornithine trans... more We performed haplotype analysis using nine single nucleotide polymorphisms in the ornithine transcarbamylase gene to explore the ancestral origins of three mutations associated with late-onset phenotype in male patients: p.R40H, p.R277W and p.Y55D. Overall, 8 haplotypes were defined among 14 families carrying p.R40H, 5 families carrying p.R277W and 2 families with p.Y55D mutations. Of nine Japanese families carrying p.R40H, eight exhibited haplotype (HT)1, whereas the other family harbored HT2. Among three Caucasian families, one Spanish and one Australian family bore HT3; one Austrian family had HT4. Two US patients harbored HT2 and HT4. Among families carrying p.R277W, HT5 was found in one Japanese, one Korean and one US family. Two other US families had HT2 and HT6. Two families carrying p.Y55D, both Japanese, shared HT1. These results indicate that the p.R40H mutation has arisen recurrently in all populations studied, although there is evidence for a founder effect in Japan, with most cases probably sharing a common origin, and to a lesser extent in subjects of European ancestry (HT3). It is evident that p.R277W mutation has recurred in discrete populations. The p.Y55D mutation appears to have arisen from a common ancestor, because this transversion (c.163T4G) occurs rarely.

Research paper thumbnail of Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures

Journal of Histochemistry & Cytochemistry, 1990

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a ke... more Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.

Research paper thumbnail of Activation of carbamoyl phosphate synthetase from Escherichia coli by glycerol

Biochemical and Biophysical Research Communications, 1982

Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryopro... more Carbamoyl phosphate synthetase I (E.C.6.3.4.16) from rat liver is activated by a range of cryoprotectants. Their diverse chemical structure and the normal stoichiometry andpattern of positional isotopic exchange for the reaction catalyzed in their presence strongly supports an allosteric mechanism for activation by the physiological activator, acetylglutamate, and by the cryoprotectants. Activation appears not to require any specific group in the cryoprotectants, but different agents activate to different degrees. All cryoprotectants found to activate are good hydrogen-bond formers and have low molecular weight (<1 000 for polyethylene glycols). This suggests that the cryoprotectant must associate with the enzyme to activate. In the presence of acetylglutamate all cryoprotectants tested, irrespective of their capacity to activate, inhibit. Carbamoyl phosphate synthetase I has activity in the absence of cryoprotectants and acetylglutamate. Under these conditions the Km's for ATP and K + are very high, the K m for NH + is very low, the Vma x appears not to exceed 15% of that at saturation of acetylglutamate and the ratio of ATPase to carbamoyl phosphate synthetase activity is ca. three times that of the acetylglutamate activated enzyme. Acetylglutamate lowers the Km's for ATP and K +, and increases the K m for NH + and the Vma x. From steady state kinetics, pulse-chase, and measurements of the rate of activation, an allosteric model for activation of the enzyme by acetylglutamate is proposed in which there are two active species, R and R • AG (AG = acetylglutamate), which have similar but not identical activities. One molecule of ATP binds with high affinity to active and inactive enzyme species, but K + and the other molecule of ATP bind preferentially to the active species. In the absence of acetylglutamate, glycerol (an effective activator) reduces the Km's for ATP and K +, increases the Vma x, and dramatically reduces the S0. 5 for acetylglutamate to <1 #M for the synthetase reaction. In the presence of excess acetylglutamate, glycerol markedly reduces the K m for ATP for the synthetase, decreases the Vmax, NH + becomes a more effective activator than K +, and Na + activates appreciably. From these and other kinetic measurements on the ATPase activity, the partial reverse reaction and rates of activation, and from pulse chase observations, the effect of glycerol is rationalized in terms of the allosteric model proposed for the actions of acetylglutamate if: a) glycerol reduces the rate of transformation and alters the allosteric equilibria between active and inactive enzyme species, and b) glycerol inhibits the active enzyme, at least in part due to slower conformational changes associated with dissociation of the products and binding of substrates. Glycerol also activates carbamoyl phosphate synthetase from E. coli in the absence of ornithine. This and the above findings are compared with data on the actions of cryoprotectants on carbamoyl phosphate synthetase II (EC 6.3.5.5) from rat liver. There are considerable similarities in the actions of these agents on the three enzymes. The relationship between these findings and the actions of cryoprotectants on other enzymes and possible physiological implications are discussed.

Research paper thumbnail of P5CS expression study in a new family with ALDH18A1‐associated hereditary spastic paraplegia SPG9

Annals of Clinical and Translational Neurology, 2019

In 2015–2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SP... more In 2015–2016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease‐causing mechanisms. We now describe a baculovirus–insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease‐causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits.

Research paper thumbnail of Insight into vitamin B6-dependent epilepsy due toPLPBP(previouslyPROSC) missense mutations

Human Mutation, 2018

Vitamin B 6-dependent genetic epilepsy was recently associated to mutations in PLPBP (previously ... more Vitamin B 6-dependent genetic epilepsy was recently associated to mutations in PLPBP (previously PROSC), the human version of the widespread COG0325 gene that encodes TIM-barrel-like pyridoxal phosphate (PLP)-containing proteins of unclear function. We produced recombinantly, purified and characterized human PROSC (called now PLPHP) and its six missense mutants reported in epileptic patients. Normal PLPHP is largely a monomer with PLP bound through a Schiff-base linkage. The PLP-targeting antibiotic Dcycloserine decreased the PLP-bound peak as expected for pseudo-first-order reaction. The p.Leu175Pro mutation grossly misfolded PLPHP. Mutations p.Arg241Gln and p.Pro87Leu decreased protein solubility and yield of pure PLPHP, but their pure forms were well folded, similarly to pure p.Pro40Leu, p.Tyr69Cys and p.Arg205Gln mutants (judged from CD spectra). PLPHP stability was decreased in p.Arg241Gln, p.Pro40Leu and p.Arg205Gln mutants (thermofluor assays). The p.Arg241Gln and p.Tyr69Cys mutants respectively lacked PLP or had a decreased amount of this cofactor. With p.Tyr69Cys there was extensive protein dimerization due to disulfide bridge formation, and PLP accessibility was decreased (judged from D-cycloserine reaction). A 3-D model of human PLPHP allowed rationalizing the effects of most mutations. Overall, the six missense mutations caused ill effects and five of them impaired folding or decreased stability, suggesting the potential of pharmacochaperonebased therapeutic approaches.

Research paper thumbnail of PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

Frontiers in microbiology, 2017

Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyan... more Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essentia...

Research paper thumbnail of Happy microbes in hostile niches. A symposium on extremophiles

International Microbiology, Mar 1, 2004

Happy microbes in hostile niches. A symposium on extremophiles Belgium can boast the privilege of... more Happy microbes in hostile niches. A symposium on extremophiles Belgium can boast the privilege of having had a microbiologist Nobel laureate (Physiology or Medicine, 1919), Jules-Jean-Baptiste-Vincent Bordet. Today, the country's outstanding microbiological tradition remains alive and well at the Research Institute of the CERIA/COOVI (1 Av. Emile Gryzon, Brussels), the center where Nicolas Glansdorff has worked for most of his scientific career and of which he was director (Fig. 1). The French-speaking-community moiety of this institute is now named Institut de Recherche Jean Marie Wiame, to honor its founding father, who was also the scientific mentor of Nicolas Glansdorff. Werner Maas (New York University) and Luigi Gorini (Harvard University) were Glansdorff's mentors in the United States. Happy Microbes, convened to honor the officially retiring Nicolas Glansdorff, paid tribute to his paramount interests in both extremophiles and the origin of life. The meeting was held on September 27, 2003, under the auspices of the Belgian Society for Microbiology, in the Etterbeek Campus

Research paper thumbnail of ALDH18A1gene mutations cause dominant spastic paraplegia SPG9: loss of function effect and plausibility of a dominant negative mechanism

Brain, 2015

Sir, We have read with great interest the article published recently in Brain by Coutelier et al.... more Sir, We have read with great interest the article published recently in Brain by Coutelier et al. (2015) 'Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia'. In support of the message of this paper, we would like to report that mutations in ALDH18A1, the causative gene in Coutelier's paper, are the cause of SPG9 (MIM#601162), a rare form of autosomal dominant hereditary spastic paraplegia (HSP) complicated with vomiting and congenital bilateral cataracts

Research paper thumbnail of SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules

Surface plasmon resonance monitoring of the binding of transcription factors CRP and NtcA from Sy... more Surface plasmon resonance monitoring of the binding of transcription factors CRP and NtcA from Synechocystis sp. PCC6803 to promoter fragments of glnA, glnN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity

Research paper thumbnail of Suggested guidelines for the diagnosis and management of urea cycle disorders

Orphanet Journal of Rare Diseases, 2012

Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to... more Urea cycle disorders (UCDs) are inborn errors of ammonia detoxification/arginine synthesis due to defects affecting the catalysts of the Krebs-Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial ornithine/citrulline antiporter) with an estimated incidence of 1:8.000. Patients present with hyperammonemia either shortly after birth (50%) or, later at any age, leading to death or to severe neurological handicap in many survivors. Despite the existence of effective therapy with alternative pathway therapy and liver transplantation, outcomes remain poor. This may be related to underrecognition and delayed diagnosis due to the nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity. These guidelines aim at providing a trans-European consensus to: guide practitioners, set standards of care and help awareness campaigns. To achieve these goals, the guidelines were developed using a Delphi methodology, by having professionals on UCDs across seven European countries to gather all the existing evidence, score it according to the SIGN evidence level system and draw a series of statements supported by an associated level of evidence. The guidelines were revised by external specialist consultants, unrelated authorities in the field of UCDs and practicing pediatricians in training. Although the evidence degree did hardly ever exceed level C (evidence from non-analytical studies like case reports and series), it was sufficient to guide practice on both acute and chronic presentations, address diagnosis, management, monitoring, outcomes, and psychosocial and ethical issues. Also, it identified knowledge voids that must be filled by future research. We believe these guidelines will help to: harmonise practice, set common standards and spread good practices with a positive impact on the outcomes of UCD patients.

Research paper thumbnail of Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine

Protein Science, 2008

The enzymes carbamoyl phosphate synthetase~CPS! and carbamate kinase~CK! make carbamoyl phosphate... more The enzymes carbamoyl phosphate synthetase~CPS! and carbamate kinase~CK! make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open b-sheet surrounded by a-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.