Golgi organization is a determinant of stem cell function in the small intestine.

Cell-to-cell signalling between niche and stem cells regulates tissue regeneration. While the identity of many mediating factors is known, it is largely unknown whether stem cells optimize their receptiveness to niche signals according to the niche organization. Here, we show that Lgr5+ small intestinal stem cells (ISCs) regulate the morphology and orientation of their secretory apparatus to match the niche architecture, and to increase transport efficiency of niche signal receptors. Unlike the progenitor cells lacking lateral niche contacts, ISCs orient Golgi apparatus laterally towards Paneth cells of the epithelial niche, and divide Golgi into multiple stacks reflecting the number of Paneth cell contacts. Stem cells with a higher number of lateral Golgi transported Epidermal growth factor receptor (Egfr) with a higher efficiency than cells with one Golgi. The lateral Golgi orientation and enhanced Egfr transport required A-kinase anchor protein 9 (Akap9), and was necessary for normal regenerative capacity in vitro . Moreover, reduced Akap9 in aged ISCs renders ISCs insensitive to niche-dependent modulation of Golgi stack number and transport efficiency. Our results reveal stem cell-specific Golgi complex configuration that facilitates efficient niche signal reception and tissue regeneration, which is compromised in the aged epithelium.

Cellular shape reinforces niche to stem cell signaling in the small intestine.

Niche-derived factors regulate tissue stem cells, but apart from the mechanosensory pathways, the effect of niche geometry is not well understood. We used organoids and bioengineered tissue culture platforms to demonstrate that the conical shape of Lgr5+ small intestinal stem cells (ISCs) facilitate their self-renewal and function. Inhibition of non-muscle myosin II (NM II)-driven apical constriction altered ISC shape and reduced niche curvature and stem cell capacity. Niche curvature is decreased in aged mice, suggesting that suboptimal interactions between old ISCs and their niche develop with age. We show that activation of NM IIC or physical restriction to young topology improves in vitro regeneration by old epithelium. We propose that the increase in lateral surface area of ISCs induced by apical constriction promotes interactions between neighboring cells, and the curved topology of the intestinal niche has evolved to maximize signaling between ISCs and neighboring cells.

Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo.

Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins.

Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/ß-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of ß-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.

Emerging candidate treatment strategies for Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is an extremely rare premature aging disorder affecting children, with a disease incidence of ∼1 in 18 million individuals. HGPS is usually caused by a de novo point mutation in exon 11 of the LMNA gene (c.1824C>T, p.G608G), resulting in the increased usage of a cryptic splice site and production of a truncated unprocessed lamin A protein named progerin. Since the genetic cause for HGPS was published in 2003, numerous potential treatment options have rapidly emerged. Strategies to interfere with the post-translational processing of lamin A, to enhance progerin clearance, or directly target the HGPS mutation to reduce the progerin-producing alternative splicing of the LMNA gene have been developed. Here, we give an up-to-date resume of the contributions made by our and other research groups to the growing list of different candidate treatment strategies that have been tested, both in vitro, in vivo in mouse models for HGPS and in clinical trials in HGPS patients.

Rare progerin-expressing preadipocytes and adipocytes contribute to tissue depletion over time.

Accumulation of progerin is believed to underlie the pathophysiology of Hutchinson-Gilford progeria syndrome, a disease characterized by clinical features suggestive of premature aging, including loss of subcutaneous white adipose tissue (sWAT). Although progerin has been found in cells and tissues from apparently healthy individuals, its significance has been debated given its low expression levels and rare occurrence. Here we demonstrate that sustained progerin expression in a small fraction of preadipocytes and adipocytes of mouse sWAT (between 4.4% and 6.7% of the sWAT cells) results in significant tissue pathology over time, including fibrosis and lipoatrophy. Analysis of sWAT from mice of various ages showed senescence, persistent DNA damage and cell death that preceded macrophage infiltration, and systemic inflammation. Our findings suggest that continuous progerin expression in a small cell fraction of a tissue contributes to aging-associated diseases, the adipose tissue being particularly sensitive.

Skin Disease in Laminopathy-Associated Premature Aging.

The nuclear lamina, a protein network located under the nuclear membrane, has during the past decade found increasing interest due to its significant involvement in a range of genetic diseases, including the segmental premature aging syndromes Hutchinson-Gilford progeria syndrome, restrictive dermopathy, and atypical Werner syndrome. In this review we examine these diseases, some caused by mutations in the LMNA gene, and their skin disease features. Advances within this area might also provide novel insights into the biology of skin aging, as recent data suggest that low levels of progerin are expressed in unaffected individuals and these levels increase with aging.

Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.

Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase.

NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+-dependent oxidation of ManNAc (N-acetyl-D-mannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH 9.4) with a high thermal stability in glycine buffer (Tm=64°C) and a strict selectivity towards ManNAc and NAD+. Crystal structures of ligand-free and ManNAc- and NAD+-bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3ß7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences.

New insights into the phylogeny and molecular classification of nicotinamide mononucleotide deamidases.

Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD(+) by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a K m value of 0.18 mM and a kcat/K m of 2.1 mM(-1) s(-1). To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases.

Insights into the evolution of sorbitol metabolism: phylogenetic analysis of SDR196C family.

BACKGROUND: Short chain dehydrogenases/reductases (SDR) are NAD(P)(H)-dependent oxidoreductases with a highly conserved 3D structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. The SDR196C family (http://www.sdr-enzymes.org) groups bacterial sorbitol dehydrogenases (SDH), which are of great industrial interest. In this study, we examine the phylogenetic relationship between the members of this family, and based on the findings and some sequence conserved blocks, a new and a more accurate classification is proposed. RESULTS: The distribution of the 66 bacterial SDH species analyzed was limited to Gram-negative bacteria. Six different bacterial families were found, encompassing α-, ß- and γ-proteobacteria. This broad distribution in terms of bacteria and niches agrees with that of SDR, which are found in all forms of life. A cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the SDH gene is surrounded by sugar ABC transporter proteins, another SDR, a kinase, and several gene regulators.According to the obtained trees, six different lineages and three sublineages can be discerned. The phylogenetic analysis also suggested two different origins for SDH in ß-proteobacteria and four origins for γ-proteobacteria.Finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for SDR and some specific blocks of SDH, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages. CONCLUSION: SDH distribution agrees with that observed for SDR, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. The phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3D structure. This suggests that SDH are very old and emerged early during the evolution. This study also opens up a new and more accurate classification of SDR196C family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, SDR196C6.3.

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences.

Characterization of a novel N-acetylneuraminate lyase from Staphylococcus carnosus TM300 and its application to N-acetylneuraminic acid production.

The possibility of incorporating N-acetylneuraminic acid (Neu5Ac) in infant formulas and other functional foods has opened up the need to synthesize N-acetylneuraminic acid using N-acetylneuraminate lyases (NALs) by reversible aldol condensation of pyruvate and N-acetyl-d-mannosamine. Until now, NALs have been cloned from pathogenic microorganisms; however, this Article describes the expression and characterization of an N-acetylneuraminate lyase from the Staphylococcus carnosus TM300, a GRAS microorganism used in fermented meat. ScNAL showed a high level of expression in E. coli (403 mg L(-1) culture). This, combined with its simple two-step purification procedure, the highest recovery described to date (86%), its kinetic parameters, which are in the same order of magnitude as best reported NALs, and its optimum pH and temperature, make ScNAL a promising and cheap biocatalyst. To confirm its biotechnological potential, the Neu5Ac was synthesized in 3 h in simple industrial working conditions with a high degree of conversion (94%).

Properties of BoAGE2, a second N-acetyl-D-glucosamine 2-epimerase from Bacteroides ovatus ATCC 8483.

N-Acyl-D-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 µM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 °C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed.

New stabilized FastPrep-CLEAs for sialic acid synthesis.

N-acetyl-D-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-D-neuraminic acid (sialic acid) from N-acetyl-D-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K(M) for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.

Molecular characterization of a novel N-acetylneuraminate lyase from Lactobacillus plantarum WCFS1.

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-d-mannosamine (ManNAc). In nature, N-acetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter(-1) culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.