4 research outputs found
Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation
Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered
Probing Multivalent Lectin-Carbohydrate Binding via Multifunctional Glycan-Gold Nanoparticles: Implications for Blocking Virus Infection
Multivalent lectin-glycan interactions are widespread
in biology and are often exploited by pathogens to bind and infect host cells.
Glycoconjugates can block such interactions and thereby prevent infection. The
inhibition potency strongly depends on matching the spatial arrangement between
the multivalent binding partners. However, the structural details of some key
lectins remain unknown and different lectins may exhibit overlapping glycan
specificity. This makes it difficult to design a glycoconjugate that can
potently and specifically target a particular multimeric lectin for therapeutic
interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon
resonance (SPR) and isothermal titration calorimetry (ITC) can provide
quantitative binding thermodynamics and kinetics. However, they cannot reveal
key structural information, e.g.
lectin’s binding site orientation, binding mode, and inter-binding site
spacing, which are critical to design specific
multivalent inhibitors. Herein we report that gold nanoparticles (GNPs)
displaying a dense layer of simple glycans are powerful mechanistic probes for
multivalent lectin-glycan interactions. They can not only quantify the
GNP-glycan-lectin binding affinities via
a new fluorescence quenching method, but also reveal drastically different
affinity enhancing mechanisms between two closely-related tetrameric lectins,
DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (inter-crosslinking with
multiple GNPs), via a combined
hydrodynamic size and electron microscopy analysis. Moreover, a new term,
potential of assembly formation (PAF) has been proposed to successfully predict
the assembly outcomes based on the binding mode between GNP-glycans and lectins.
Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated
augmentation of Ebola virus glycoprotein-driven cell entry (with IC50
values down to 95 pM), but only partially block DC-SIGNR-mediated virus
infection. Our results suggest that the ability of a glycoconjugate to
simultaneously block all binding sites of a target lectin is key to robust
inhibition of viral infection
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Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly
Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase