Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include ...craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.
The crystal structure of UDP‐N‐acetylglucosamine 4‐epimerase (UDP‐GlcNAc 4‐epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a ...resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N‐terminal Rossmann‐fold for nucleotide binding and an active site positioned in the C‐terminus. UDP‐GlcNAc 4‐epimerase is a member of the short‐chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4‐epimerases. The protein was co‐crystallized with coenzyme NADH and UDP‐N‐acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non‐native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme.
Methyl‐coenzyme M reductase (MCR) is a multi‐subunit (α2β2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are ...typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high‐resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin‐like domain assembled into an α + β barrel‐like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.
Methyl‐coenzyme M reductase (MCR) is responsible for biological methanogenesis. McrD and MCR are conserved and colocated in methanogen genomes, with McrD protein able to form a complex with MCR. We present the high‐resolution McrD crystal structure of ferredoxin‐like domain monomers assembled into an α + β barrel‐like dimer, with conformational heterogeneity exhibited by a key loop. Created with BioRender.com.
Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, ...although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes—MAP3K7, encoding transforming growth factor β (TGF-β)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T p.Pro485Leu) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C p.Glu70Gln, c.299T>A p.Val100Glu, and c.502G>C p.Gly168Arg). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.
Two novel mutants in the strigolactone pathway demonstrate a role for the hormone in sepal senescence, and transcriptional analysis highlights interaction between strigolactones and sugar signalling.
...Abstract
Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem–iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.
The regulated proliferation and differentiation of neural stem cells before the generation and migration of neurons in the cerebral cortex are central aspects of mammalian development. ...Periventricular neuronal heterotopia, a specific form of mislocalization of cortical neurons, can arise from neuronal progenitors that fail to negotiate aspects of these developmental processes. Here we show that mutations in genes encoding the receptor-ligand cadherin pair DCHS1 and FAT4 lead to a recessive syndrome in humans that includes periventricular neuronal heterotopia. Reducing the expression of Dchs1 or Fat4 within mouse embryonic neuroepithelium increased progenitor cell numbers and reduced their differentiation into neurons, resulting in the heterotopic accumulation of cells below the neuronal layers in the neocortex, reminiscent of the human phenotype. These effects were countered by concurrent knockdown of Yap, a transcriptional effector of the Hippo signaling pathway. These findings implicate Dchs1 and Fat4 upstream of Yap as key regulators of mammalian neurogenesis.
The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, ...utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the “open” conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal ...actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.
Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens
Methanobacteria
and
Methanopyrus
possess cell walls of pseudomurein, a structural ...analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar
N
-acetyltalosaminuronic acid instead of
N
-acetylmuramic acid, β−1,3 glycosidic bonds in place of β−1,4 bonds and only
l
-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from
Methanothermus fervidus
(Mfer762) and
Methanothermobacter thermautotrophicus
(Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.
Bioengineered polyhydroxyalkanoate (PHA) spheres assembled in engineered bacteria are showing promising potential in protein immobilization for high-value applications. Here, we have designed ...innovative streamlined approaches to add functional proteins from complex mixtures (e.g., without prior purification) to bioengineered PHA spheres directly harnessing the specificity of the SpyTag/SpyCatcher mediated protein ligation.
was engineered to assemble PHA spheres displaying the SpyCatcher domain while simultaneously producing a SpyTagged target protein, which was
specifically ligated to the PHA spheres. To further demonstrate the specificity of this ligation reaction, we incubated isolated SpyCatcher-coated PHA spheres with cell lysates containing SpyTagged target protein, which also resulted in specific ligation mediating surface functionalization. An even cruder approach was used by lysing a mixture of cells, either producing PHA spheres or target protein, which resulted in specific surface functionalization suggesting that ligation between the SpyCatcher-coated PHA spheres and the SpyTagged target proteins is highly specific. To expand the design space of this general modular approach toward programmable multifunctionalization, e.g., one-pot construction of immobilized multienzyme cascade systems on PHA spheres, we designed various recombinant bimodular PHA spheres utilizing alternative Tag/Catcher pairs (e.g., SnoopTag/SnoopCatcher and SdyTag/SdyCatcher systems). One of our bimodular PHA spheres resulted in simultaneous multifunctionalization of plain PHA spheres in one-step with two differently tagged proteins under
and
reaction conditions while remaining functional. Our bimodular PHA spheres also showed high orthogonality with the non-target peptide tag and exhibited decent robustness against repeated freeze-thaw treatment. We demonstrated the utility of these approaches by using a fluorescent protein, a monomeric amylase, and a dimeric organophosphate hydrolase as target proteins. We established a versatile toolbox for dynamic functionalization of PHA spheres for biomedical and industrial applications.
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