How cells dedifferentiate: a lesson from plants

The remarkable regenerative capacity displayed by plants and various vertebrates, such as amphibians, is largely based on the capability of somatic cells to undergo dedifferentiation. In this process, mature cells reverse their state of differentiation and acquire pluripotentiality--a process preceding not only reentry into the cell cycle but also a commitment for cell death or trans- or redifferentiation. Recent studies provide a new perspective on cellular dedifferentiation, establishing chromatin reorganization as its fundamental theme.     

The SMN complex

The survival of motor neurons (SMN) protein is the product of the disease-determining gene of the neurodegenerative disorder spinal muscular atrophy (SMA). SMN is part of a stable multiprotein complex that is found in all metazoan cells in the cytoplasm and in nuclear Gems. The SMN complex contains, in addition to SMN, at least six other proteins, named Gemins2-7, and plays an essential role in the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs). Through its binding to specific sequences in the snRNAs, the SMN complex surveys the correct identity of the target RNAs and facilitates snRNP assembly. Based on its ability to interact with several other protein and RNA components of cellular RNPs, it is likely that the SMN complex functions as an assemblyosome in the formation of diverse RNP particles, some of which may be of particular importance to the motor neuron. A detailed understanding of the cellular roles of the SMN complex may help the development of therapeutic strategies for this neurodegenerative disease.     

Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.     

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.     

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.     

PLA2G6 mutation underlies infantile neuroaxonal dystrophy

Infantile neuroaxonal dystrophy (INAD) is an autosomal recessive progressive neurodegenerative disease that presents within the first 2 years of life and culminates in death by age 10 years. Affected individuals from two unrelated Bedouin Israeli kindreds were studied. Brain imaging demonstrated diffuse cerebellar atrophy and abnormal iron deposition in the medial and lateral globus pallidum. Progressive white-matter disease and reduction of the N-acetyl aspartate : chromium ratio were evident on magnetic resonance spectroscopy, suggesting loss of myelination. The clinical and radiological diagnosis of INAD was verified by sural nerve biopsy. The disease gene was mapped to a 1.17-Mb locus on chromosome 22q13.1 (LOD score 4.7 at recombination fraction 0 for SNP rs139897), and an underlying mutation common to both affected families was identified in PLA2G6, the gene encoding phospholipase A2 group VI (cytosolic, calcium-independent). These findings highlight a role of phospholipase in neurodegenerative disorders.     

Gene expression of Caenorhabditis elegans neurons carries information on their synaptic connectivity

The claim that genetic properties of neurons significantly influence their synaptic network structure is a common notion in neuroscience. The nematode Caenorhabditis elegans provides an exciting opportunity to approach this question in a large-scale quantitative manner. Its synaptic connectivity network has been identified, and, combined with cellular studies, we currently have characteristic connectivity and gene expression signatures for most of its neurons. By using two complementary analysis assays we show that the expression signature of a neuron carries significant information about its synaptic connectivity signature, and identify a list of putative genes predicting neural connectivity. The current study rigorously quantifies the relation between gene expression and synaptic connectivity signatures in the C. elegans nervous system and identifies subsets of neurons where this relation is highly marked. The results presented and the genes identified provide a promising starting point for further, more detailed computational and experimental investigations.     

Mechanisms of initial endplate failure in the human vertebral body

Endplate failure occurs frequently in osteoporotic vertebral fractures and may be related to the development of high tensile strain. To determine whether the highest tensile strains in the vertebra occur in the endplates, and whether such high tensile strains are associated with the material behavior of the intervertebral disc, we used micro-CT-based finite element analysis to assess tissue-level strains in 22 elderly human vertebrae (81.5±9.6 years) that were compressed through simulated intervertebral discs. In each vertebra, we compared the highest tensile and compressive strains across the different compartments: endplates, cortical shell, and trabecular bone. The influence of Poisson-type expansion of the disc on the results was determined by compressing the vertebrae a second time in which we suppressed the Poisson expansion. We found that the highest tensile strains occurred within the endplates whereas the highest compressive strains occurred within the trabecular bone. The ratio of strain to assumed tissue-level yield strain was the highest for the endplates, indicating that the endplates had the greatest risk of initial failure. Suppressing the Poisson expansion of the disc decreased the amount of highly tensile-strained tissue in the endplates by 79.4±11.3%. These results indicate that the endplates are at the greatest risk of initial failure due to the development of high tensile strains, and that such high tensile strains are associated with the Poisson expansion of the disc. We conclude that initial failure of the vertebra is associated with high tensile strains in the endplates, which in turn are influenced by the material behavior of the disc.     

Enantioselective metabolism of ifosfamide by the kidney

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Enantioselective oxidation of IF has been shown in the liver but has never been reported in the kidney. Using porcine and human kidney samples, as well as the renal porcine cell line LLCPK-1, we document enantioselective metabolism of IF with prevalent production of the N-dechloroethylifosfamide (DCEIF) metabolites from the (S)-IF enantiomer compared to the amount of N-DCEIF metabolites produced from the (R)-IF enantiomers. Since IF enantiomers appear to be equally effective in chemotherapy, these results suggest that replacing the clinically standard racemic mixture of IF with (R)-IF may decrease renal metabolism of the drug and hence may decrease nephrotoxicity.     

Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification

Glycosylation is a key mechanism for orchestrating the bioactivity, metabolism and location of small molecules in living cells. In plants, a large multigene family of glycosyltransferases is involved in these processes, conjugating hormones, secondary metabolites, biotic and abiotic environmental toxins, to impact directly on cellular homeostasis. The red grape enzyme UDP-glucose:flavonoid 3-O-glycosyltransferase (VvGT1) is responsible for the formation of anthocyanins, the health-promoting compounds which, in planta, function as colourants determining flower and fruit colour and are precursors for the formation of pigmented polymers in red wine. We show that VvGT1 is active, in vitro, on a range of flavonoids. VvGT1 is somewhat promiscuous with respect to donor sugar specificity as dissected through full kinetics on a panel of nine sugar donors. The three-dimensional structure of VvGT1 has also been determined, both in its 'Michaelis' complex with a UDP-glucose-derived donor and the acceptor kaempferol and in complex with UDP and quercetin. These structures, in tandem with kinetic dissection of activity, provide the foundation for understanding the mechanism of these enzymes in small molecule homeostasis.