Viet Q. Le

Structure of bone morphogenetic protein 9 procomplex.

Significance Bone morphogenetic protein (BMP) activity is regulated by prodomains. Here, structures of BMP procomplexes reveal an open-armed conformation. In contrast, the evolutionarily related, latent TGF-β1 procomplex is cross-armed. We propose that in the TGF-β and BMP family, conversion between cross-armed and open-armed conformations may regulate release and activity of the growth factor. Bone morphogenetic proteins (BMPs) belong to the TGF-β family, whose 33 members regulate multiple aspects of morphogenesis. TGF-β family members are secreted as procomplexes containing a small growth factor dimer associated with two larger prodomains. As isolated procomplexes, some members are latent, whereas most are active; what determines these differences is unknown. Here, studies on pro-BMP structures and binding to receptors lead to insights into mechanisms that regulate latency in the TGF-β family and into the functions of their highly divergent prodomains. The observed open-armed, nonlatent conformation of pro-BMP9 and pro-BMP7 contrasts with the cross-armed, latent conformation of pro-TGF-β1. Despite markedly different arm orientations in pro-BMP and pro-TGF-β, the arm domain of the prodomain can similarly associate with the growth factor, whereas prodomain elements N- and C-terminal to the arm associate differently with the growth factor and may compete with one another to regulate latency and stepwise displacement by type I and II receptors. Sequence conservation suggests that pro-BMP9 can adopt both cross-armed and open-armed conformations. We propose that interactors in the matrix stabilize a cross-armed pro-BMP conformation and regulate transition between cross-armed, latent and open-armed, nonlatent pro-BMP conformations.

Nuclear export modulates the cytoplasmic Sir2 homologue Hst2

Modulating transcription factors is crucial to executing sophisticated gene expression programs. The silent information regulator 2 (Sir2) family of NAD‐dependent protein deacetylases influences transcription by targeting proteins such as histones, p53 and forkhead‐box family transcription factors. Although apparently cytoplasmic, both mammalian SIRT2 and its yeast orthologue Hst2 have been implicated in transcriptional regulation. Here, we show that Hst2 moves between the nucleus and cytoplasm, but is largely cytoplasmic owing to efficient nuclear export. This nuclear exclusion is mediated by the exportin chromosomal region maintenance 1 (Crm1) and a putative leucine‐rich nuclear export sequence in Hst2, which overlaps a unique autoregulatory helix. Disruption of Hst2 export shows that nuclear exclusion inhibits the activity of Hst2 as a transcriptional repressor. Our identification of putative nuclear export sequences in numerous vertebrate SIRT2 proteins shows that active nuclear export can be a conserved mechanism for regulating Sir2 homologues.

Hyperactive BMP signaling induced by ALK2(R206H) requires type II receptor function in a Drosophila model for classic fibrodysplasia ossificans progressiva.

Background: Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal dominant disorder characterized by episodic deposition of heterotopic bone in place of soft connective tissue. All FOP‐associated mutations map to the BMP type I receptor, ALK2, with the ALK2R206H mutant form found in the vast majority of patients. The mechanism(s) regulating the expressivity of hyperactive ALK2R206H signaling throughout a patients life is not well understood. Results: In Drosophila, human ALK2R206H receptor induces hyperactive BMP signaling. As in vertebrates, elevated signaling associated with ALK2R206H in Drosophila is ligand‐independent. We found that a key determinant for ALK2R206H hyperactivity is a functional type II receptor. Furthermore, our results indicate that like its Drosophila ortholog, Saxophone (Sax), wild‐type ALK2 can antagonize, as well as promote, BMP signaling. Conclusions: The dual function of ALK2 is of particular interest given the heterozygous nature of FOP, as the normal interplay between such disparate behaviors could be shifted by the presence of ALK2R206H receptors. Our studies provide a compelling example for Drosophila as a model organism to study the molecular underpinnings of complex human syndromes such as FOP. Developmental Dynamics 241:200–214, 2012.

Functional Analysis of saxophone , the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-β signaling.

2, 2′- and 4, 4′-Cyanines are transporter-independent in vitro dopaminergic toxins with the specificity and mechanism of toxicity similar to MPP+

Specific uptake through dopamine transporter followed by the inhibition of the mitochondrial complex‐I have been accepted as the cause of the specific dopaminergic toxicity of 1‐methyl‐4‐phenylpyridinium (MPP+). However, MPP+ is taken up into many cell types through other transporters, suggesting that, in addition to the efficient uptake, intrinsic vulnerability of dopaminergic cells may also contribute to their high sensitivity to MPP+ and similar toxins. To test this possibility, two simple cyanines were employed in a comparative study based on their unique characteristics and structural similarity to MPP+. Here, we show that they freely accumulate in dopaminergic (MN9D and SH‐SY5Y) as well as in liver (HepG2) cells, but are specifically and highly toxic to dopaminergic cells with IC50s in the range of 50–100 nM, demonstrating that they are about 1000‐fold more toxic than MPP+ under similar experimental conditions. They cause mitochondrial depolarization non‐specifically, but increase the reactive oxygen species specifically in dopaminergic cells leading to the apoptotic cell death parallel to MPP+. These and other findings suggest that the specific dopaminergic toxicity of these cyanines is due to the inherent vulnerability of dopaminergic cells toward mitochondrial toxins that lead to the excessive production of reactive oxygen species. Therefore, the specific dopaminergic toxicity of MPP+ must also be, at least partly, due to the specific vulnerability of dopaminergic neurons. Thus, these cyanines could be stronger in vivo dopaminergic toxins than MPP+ and their in vivo toxicities must be evaluated.

Tolloid cleavage activates latent GDF8 by priming the pro‐complex for dissociation

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF‐β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid‐cleaved GDF8 pro‐complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro‐complexes reveals a V‐shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring‐like, cross‐armed conformation of latent TGF‐β1. Surprisingly, Tolloid‐cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen–deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain–growth factor dissociation, increased exchange in regions that correspond in pro‐TGF‐β1 to the α1‐helix, latency lasso, and β1‐strand in the prodomain and to the β6′‐ and β7′‐strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain–growth factor interfaces and primes the growth factor for release from the prodomain.

Gene Expression-Based Screen for Parkinson’s Disease Identifies GW8510 as a Neuroprotective Agent

We carried out a gene expression-based in silico screen in order to identify small molecules with gene-expression profiles that are anticorrelated with a gene-expression profile for Parkinsons disease (PD). We identified the cyclin-dependent kinase 2/5 (CDK2/5) inhibitor GW8510 as our most significant hit and characterized its effects in rodent MN9D cells and in human neuronal cells derived from induced pluripotent stem cells. GW8510 demonstrated neuroprotective ability in MN9D cells in the presence of 1-methyl-4-phenylpyridium (MPP(+)), a widely used neurotoxin model for Parkinsons disease. In order to delineate the nature and extent of GW8510s neuroprotective properties, we studied GW8510 in human neuronal cells in the context of various mechanisms of cellular stress. We found that GW8510 was protective against small-molecule mitochondrial and endoplasmic reticulum stressors. Our findings illustrate an approach to using small-molecule gene expression libraries to identify compounds with therapeutic potential in human diseases.