Genomic integrity and mitochondrial metabolism defects in Warsaw syndrome cells: a comparison with Fanconi anemia.

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient-derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases.

The permeation mechanism of organic cations through a CNG mimic channel.

Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+ channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA). We combined electrophysiology and molecular dynamics simulations to examine how DMA interacts with the pore and permeates through it. Due to the presence of hydrophobic groups, DMA enters easily in the channel and, unlike the alkali cations, does not need to cross any barrier. We also show that while the crystal structure is consistent with the presence of a single DMA ion at full occupancy, the channel is able to conduct a sizable current of DMA ions only when two ions are present inside the channel. Moreover, the second DMA ion dramatically changes the free energy landscape, destabilizing the crystallographic binding site and lowering by almost 25 kJ/mol the binding affinity between DMA and the channel. Based on the results of the simulation the experimental electron density maps can be re-interpreted with the presence of a second ion at lower occupancy. In this mechanism the flexibility of the channel plays a key role, extending the classical multi-ion permeation paradigm in which conductance is enhanced by the plain interaction between the ions.

The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35.

MID1/TRIM18 is a member of the TRIM family of ubiquitin E3 ligases characterized by the presence of a conserved RING-containing N-terminal tripartite motif. Mutations in the MID1 gene have been associated with the X-linked form of Opitz Syndrome, a developmental disorder characterized by midline defects and intellectual disability. The effect of MID1 E3 ligase activity within the cell and the role in the pathogenesis of the disease is still not completely unraveled. Here, we report BRAF35, a non-canonical HMG nuclear factor, as a novel MID1 substrate. MID1 is implicated in BRAF35 ubiquitination promoting atypical poly-ubiquitination via K6-, K27- and K29-linkages. We observed a partial co-localization of the two proteins within cytoplasmic bodies. We found that MID1 depletion alters BRAF35 localization in these structures and increases BRAF35 stability affecting its cytoplasmic abundance. Our data reveal a novel role for MID1 and for atypical ubiquitination in balancing BRAF35 presence, and likely its activity, within nuclear and cytoplasmic compartments.

Exploring the G-quadruplex binding and unwinding activity of the bacterial FeS helicase DinG.

Despite numerous reports on the interactions of G-quadruplexes (G4s) with helicases, systematic analysis addressing the selectivity and specificity of each helicase towards a variety of G4 topologies are scarce. Among the helicases able to unwind G4s are those containing an iron-sulphur (FeS) cluster, including both the bacterial DinG (found in E. coli and several pathogenic bacteria) and the medically important eukaryotic homologues (XPD, FancJ, DDX11 and RTEL1). We carried out a detailed study of the interactions between the E. coli DinG and a variety of G4s, by employing physicochemical and biochemical methodologies. A series of G4-rich sequences from different genomic locations (promoter and telomeric regions), able to form unimolecular G4 structures with diverse topologies, were analyzed (c-KIT1, KRAS, c-MYC, BCL2, Tel23, T30695, Zic1). DinG binds to most of the investigated G4s with little discrimination, while it exhibits a clear degree of unwinding specificity towards different G4 topologies. Whereas previous reports suggested that DinG was active only on bimolecular G4s, here we show that it is also able to bind to and resolve the more physiologically relevant unimolecular G4s. In addition, when the G4 structures were stabilized by ligands (Pyridostatin, PhenDC3, BRACO-19 or Netropsin), the DinG unwinding activity decreased and in most cases was abolished, with a pattern that is not simply explained by a change in binding affinity. Overall, these results have important implications for the biochemistry of helicases, strongly suggesting that when analysing the G4 unwinding property of an enzyme, it is necessary to investigate a variety of G4 substrates.

Lack of Mid1, the mouse ortholog of the Opitz syndrome gene, causes abnormal development of the anterior cerebellar vermis.

Opitz G/BBB syndrome (OS) is a genetic disorder characterized by midline developmental defects. Male patients with the X-linked form of OS, caused by loss-of-function mutations in the MID1 gene, show high variability of the clinical signs. MID1 encodes a ubiquitin ligase that controls phosphatase 2A, but its role in the pathogenesis of the disease is still unclear. Here, we report a mouse line carrying a nonfunctional ortholog of the human MID1 gene, Mid1. Mid1-null mice show the brain anatomical defect observed in patients (i.e., hypoplasia of the anterior portion of the medial cerebellum, the vermis). We found that the presence of this defect correlates with motor coordination and procedural and nonassociative learning impairments. The defect is limited to the most anterior lobes of the vermis, the region of the developing cerebellum adjacent to the dorsal midbrain. Analyses at midgestation reveal that lack of Mid1 causes the shortening of the posterior dorsal midbrain, the rostralization of the midbrain/cerebellum boundary, and the downregulation of a key player in the development of this region, Fgf17. Thus, lack of Mid1 causes a misspecification of the midbrain/cerebellar boundary that results in an abnormal development of the most anterior cerebellar lobes. This animal model provides a tool for additional in vivo studies of the physiological and pathological role of the Mid1 gene and a system to investigate the development and function of anterior cerebellar domains.

Two further patients with Warsaw breakage syndrome. Is a mild phenotype possible?

BACKGROUND: Warsaw Breakage Syndrome (WABS) is an ultra rare cohesinopathy caused by biallelic mutation of DDX11 gene. It is clinically characterized by pre and postnatal growth delay, microcephaly, hearing loss with cochlear hypoplasia, skin color abnormalities, and dysmorphisms. METHODS: Mutational screening and functional analyses (protein expression and 3D-modeling) were performed in order to investigate the presence and pathogenicity of DDX11 variant identified in our patients. RESULTS: We report the clinical history of two sisters affected by WABS with a pathological mytomicin C test carrying compound heterozygous mutations (c.2507T > C / c.907_920del) of the DDX11 gene. The pathogenicity of this variant was confirmed in the light of a bioinformatic study and protein three-dimensional modeling, as well as expression analysis. CONCLUSION: These findings further extend the clinical and molecular knowledge about the WABS showing a possible mild phenotype without major malformations or intellectual disability.

Williams-Beuren syndrome TRIM50 encodes an E3 ubiquitin ligase.

Williams-Beuren syndrome (WBS) is a neurodevelopmental and multisystemic disease that results from hemizygosity of approximately 25 genes mapping to chromosomal region 7q11.23. We report here the preliminary description of eight novel genes mapping within the WBS critical region and/or its syntenic mouse region. Three of these genes, TRIM50, TRIM73 and TRIM74, belong to the TRIpartite motif gene family, members of which were shown to be associated to several human genetic diseases. We describe the preliminary functional characterization of these genes and show that Trim50 encodes an E3 ubiquitin ligase, opening the interesting hypothesis that the ubiquitin-mediated proteasome pathway might be involved in the WBS phenotype.

Molecular and Cellular Functions of the Warsaw Breakage Syndrome DNA Helicase DDX11.

DDX11/ChlR1 (Chl1 in yeast) is a DNA helicase involved in sister chromatid cohesion and in DNA repair pathways. The protein belongs to the family of the iron⁻sulphur cluster containing DNA helicases, whose deficiencies have been linked to a number of diseases affecting genome stability. Mutations of human DDX11 are indeed associated with the rare genetic disorder named Warsaw breakage syndrome, showing both chromosomal breakages and chromatid cohesion defects. Moreover, growing evidence of a potential role in oncogenesis further emphasizes the clinical relevance of DDX11. Here, we illustrate the biochemical and structural features of DDX11 and how it cooperates with multiple protein partners in the cell, acting at the interface of DNA replication/repair/recombination and sister chromatid cohesion to preserve genome stability.

The gating mechanism in cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs' binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members. To date, only the open, cGMP-bound, structure of a CNG channel has been determined at atomic resolution, which is inadequate to determine the molecular events underlying gating. By using electrophysiology, site-directed mutagenesis, chemical modification, and Single Molecule Force Spectroscopy, we demonstrate that opening of CNGA1 channels is initiated by the formation of salt bridges between residues in the C-linker and S5 helix. These events trigger conformational changes of the α-helix S5, transmitted to the P-helix and leading to channel opening. Therefore, the superfamily of voltage-gated channels shares a similar molecular architecture but has evolved divergent gating mechanisms.

Actin Waves Do Not Boost Neurite Outgrowth in the Early Stages of Neuron Maturation.

During neurite development, Actin Waves (AWs) emerge at the neurite base and move up to its tip, causing a transient retraction of the Growth Cone (GC). Many studies have shown that AWs are linked to outbursts of neurite growth and, therefore, contribute to the fast elongation of the nascent axon. Using long term live cell-imaging, we show that AWs do not boost neurite outgrowth and that neurites without AWs can elongate for several hundred microns. Inhibition of Myosin II abolishes the transient GC retraction and strongly modifies the AWs morphology. Super-resolution nanoscopy shows that Myosin IIB shapes the growth cone-like AWs structure and is differently distributed in AWs and GCs. Interestingly, depletion of membrane cholesterol and inhibition of Rho GTPases decrease AWs frequency and velocity. Our results indicate that Myosin IIB, membrane tension, and small Rho GTPases are important players in the regulation of the AW dynamics. Finally, we suggest a role for AWs in maintaining the GCs active during environmental exploration.

Cdc42 and RhoA reveal different spatio-temporal dynamics upon local stimulation with Semaphorin-3A.

Small RhoGTPases, such as Cdc42 and RhoA, are key players in integrating external cues and intracellular signaling pathways that regulate growth cone (GC) motility. Indeed, Cdc42 is involved in actin polymerization and filopodia formation, whereas RhoA induces GC collapse and neurite retraction through actomyosin contraction. In this study we employed Förster Resonance Energy Transfer (FRET) microscopy to study the spatio-temporal dynamics of Cdc42 and RhoA in GCs in response to local Semaphorin-3A (Sema3A) stimulation obtained with lipid vesicles filled with Sema3A and positioned near the selected GC using optical tweezers. We found that Cdc42 and RhoA were activated at the leading edge of NG108-15 neuroblastoma cells during spontaneous cycles of protrusion and retraction, respectively. The release of Sema3A brought to a progressive activation of RhoA within 30 s from the stimulus in the central region of the GC that collapsed and retracted. In contrast, the same stimulation evoked waves of Cdc42 activation propagating away from the stimulated region. A more localized stimulation obtained with Sema3A coated beads placed on the GC, led to Cdc42 active waves that propagated in a retrograde manner with a mean period of 70 s, and followed by GC retraction. Therefore, Sema3A activates both Cdc42 and RhoA with a complex and different spatial-temporal dynamics.