Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/ gelatin-binding region between domains FNI 6 and FNI 9 . INTRODUCTIONFibrillins are extracellular matrix components with important functions in elastic and nonelastic tissues including blood vessels, bone, and the eye. Fibrillins, together with the latent transforming growth factor-␤ (TGF-␤)-binding proteins (LTBPs), constitute the fibrillin-LTBP family of proteins (Hubmacher et al., 2006). Fibrillins are ϳ350 kDa in size, are disulfide-rich and glycosylated, and have a characteristic modular structure. The three human fibrillinsfibrillin-1, -2, and -3 -are encoded by different genes and are conserved at the amino acid level both in relation to one another and among species. Fibrillins are mainly composed of tandem arrays of calcium-binding epidermal growth factor-like domains interspersed with TGF-␤-binding protein domains (TB/8-Cys) and hybrid domains Hubmacher et al., 2006). Mutations in fibrillins give rise to the so-called fibrillinopathies, which include Marfan syndrome and autosomal dominant Weill-Marchesani syndrome, both caused by mutations in fibrillin-1, and Beal's syndrome, caused by mutations in fibrillin-2 (Robinson et al., 2006).High-molecular-weight, multiprotein assemblies called microfibrils are the functional units of fibrillins, which serve as a scaffold for the biogenesis of elastic fibers, confer structural integrity to individual organ systems, regulate growth factor signaling of TGF-␤-bone morphogenic protein (BMP) superfamily members and provide limited elasticity to tissues (Kielty et al., 2002;Charbonneau et al., 2004;Ramirez and Dietz, 2007). Extracted microfibrils from cell culture or tissues display a typical bead-on-a-string ultrastructure, having a 50 -55-nm periodicity when analyzed by ele...

BackgroundNeurodevelopmental disorders have challenged clinical genetics for decades, with over 700 genes implicated and many whose function remains unknown. The application of whole-exome sequencing is proving pivotal in closing the genotype/phenotype gap through the discovery of new genes and variants that help to unravel the pathogenic mechanisms driving neuropathogenesis. One such discovery includes TRIO, a gene recently implicated in neurodevelopmental delay. Trio is a Dbl family guanine nucleotide exchange factor (GEF) and a major regulator of neuronal development, controlling actin cytoskeleton dynamics by activating the GTPase Rac1.MethodsWhole-exome sequencing was undertaken on a family presenting with global developmental delay, microcephaly and mild dysmorphism. Father/daughter exome analysis was performed, followed by confirmatory Sanger sequencing and segregation analysis on four individuals. Three further patients were recruited through the deciphering developmental disorders (DDD) study. Functional studies were undertaken using patient-specific Trio protein mutations.ResultsWe identified a frameshift deletion in TRIO that segregated autosomal dominantly. By scrutinising data from DDD, we further identified three unrelated children with a similar phenotype who harboured de novo missense mutations in TRIO. Biochemical studies demonstrated that in three out of four families, the Trio mutations led to a markedly reduced Rac1 activation.ConclusionsWe describe an inherited global developmental delay phenotype associated with a frameshift deletion in TRIO. Additionally, we identify pathogenic de novo missense mutations in TRIO associated with the same consistent phenotype, intellectual disability, microcephaly and dysmorphism with striking digital features. We further functionally validate the importance of the GEF domain in Trio protein function. Our study demonstrates how genomic technologies are yet again proving prolific in diagnosing and advancing the understanding of neurodevelopmental disorders.

The Rho-guanine nucleotide exchange factor (RhoGEF) TRIO acts as a key regulator of neuronal migration, axonal outgrowth, axon guidance, and synaptogenesis by activating the GTPase RAC1 and modulating actin cytoskeleton remodeling. Pathogenic variants in TRIO are associated with neurodevelopmental diseases, including intellectual disability (ID) and autism spectrum disorders (ASD). Here, we report the largest international cohort of 24 individuals with confirmed pathogenic missense or nonsense variants in TRIO. The nonsense mutations are spread along the TRIO sequence, and affected individuals show variable neurodevelopmental phenotypes. In contrast, missense variants cluster into two mutational hotspots in the TRIO sequence, one in the seventh spectrin repeat and one in the RAC1-activating GEFD1. Although all individuals in this cohort present with developmental delay and a neuro-behavioral phenotype, individuals with a pathogenic variant in the seventh spectrin repeat have a more severe ID associated with macrocephaly than do most individuals with GEFD1 variants, who display milder ID and microcephaly. Functional studies show that the spectrin and GEFD1 variants cause a TRIO-mediated hyper-or hypo-activation of RAC1, respectively, and we observe a striking correlation between RAC1 activation levels and the head size of the affected individuals. In addition, truncations in TRIO GEFD1 in the vertebrate model X. tropicalis induce defects that are concordant with the human phenotype. This work demonstrates distinct clinical and molecular disorders clustering in the GEFD1 and seventh spectrin repeat domains and highlights the importance of tight control of TRIO-RAC1 signaling in neuronal development.

Defects in protein folding and the proteasomal pathway have been linked with many neurodegenerative diseases. PLIC-1 (protein linking IAP to the cytoskeleton) is a ubiquitin-like protein that binds to the ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a. Here, we show that PLIC-1 also binds to the UIM proteins ataxin 3-a deubiquitinating enzymeHSJ1a-a co-chaperone-and EPS15 (epidermal growth factor substrate 15)-an endocytic protein. Using a polyglutamine (polyQ) disease model, we found that both endogenous PLIC-1 and EPS15 localize to perinuclear aggresomes, and that polyQ enhances their in vivo interaction. We show that knockdown of PLIC-1 and EPS15 by RNA interference reduces aggresome formation. In addition, PLIC-1 DUBL functions as a dominantnegative mutant, blocking both polyQ transport to aggresomes and the association of EPS15 with dispersed aggregates. We also show that PLIC-1 is upregulated by arsenite-induced protein misfolding. These results indicate a role for PLIC-1 in the protein aggregation-stress pathway, and we propose a novel function for the ubiquitin-like (UBL) domain-by means of UBL-UIM interactions-in transport to aggresomes.

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.