The prodomain of bone morphogenetic protein 2 promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus. We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

The anti-Müllerian hormone prodomain is displaced from the hormone/prodomain complex upon bivalent binding to the hormone receptor.

Noncovalent complexes of transforming growth factor-ß family growth/differentiation factors with their prodomains are classified as latent or active, depending on whether the complexes can bind their respective receptors. For the anti-Müllerian hormone (AMH), the hormone-prodomain complex is active, and the prodomain is displaced upon binding to its type II receptor, AMH receptor type-2 (AMHR2), on the cell surface. However, the mechanism by which this displacement occurs is unclear. Here, we used ELISA assays to measure the dependence of prodomain displacement on AMH concentration and analyzed results with respect to the behavior expected for reversible binding in combination with ligand-induced receptor dimerization. We found that, in solution, the prodomain has a high affinity for the growth factor (GF) (Kd = 0.4 pM). Binding of the AMH complex to a single AMHR2 molecule does not affect this Kd and does not induce prodomain displacement, indicating that the receptor binding site in the AMH complex is fully accessible to AMHR2. However, recruitment of a second AMHR2 molecule to bind the ligand bivalently leads to a 1000-fold increase in the Kd for the AMH complex, resulting in rapid release of the prodomain. Displacement occurs only if the AMHR2 is presented on a surface, indicating that prodomain displacement is caused by a conformational change in the GF induced by bivalent binding to AMHR2. In addition, we demonstrate that the bone morphogenetic protein 7 prodomain is displaced from the complex with its GF by a similar process, suggesting that this may represent a general mechanism for receptor-mediated prodomain displacement in this ligand family.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-ß precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

Structure of the human myostatin precursor and determinants of growth factor latency.

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-ß1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors.

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

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-ß 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-ß1. 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-ß1 to the α1-helix, latency lasso, and ß1-strand in the prodomain and to the ß6'- and ß7'-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.

The role of pro-domains in human growth factors and cytokines.

Many growth factors and cytokines are produced as larger precursors, containing pro-domains, that require proteolytic processing to release the bioactive ligand. These pro-domains can be significantly larger than the mature domains and can play an active role in the regulation of the ligands. Mining the UniProt database, we identified almost one hundred human growth factors and cytokines with pro-domains. These are spread across several unrelated protein families and vary in both their size and composition. The precise role of each pro-domain varies significantly between the protein families. Typically they are critical for controlling bioactivity and protein localisation, and they facilitate diverse mechanisms of activation. Significant gaps in our understanding remain for pro-domain function - particularly their fate once the bioactive ligand has been released. Here we provide an overview of pro-domain roles in human growth factors and cytokines, their processing, regulation and activation, localisation as well as therapeutic potential.

Molecular characterization of latent GDF8 reveals mechanisms of activation.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-ß. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-ß and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Prodomain-growth factor swapping in the structure of pro-TGF-ß1.

TGF-ß is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-ß is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked before proprotein convertase cleavage and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-ß1 with the proprotein convertase cleavage site mutated to mimic the structure of the TGF-ß1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure (i.e. the prodomain arm domain and GF domain in each monomer are swapped). Swapping has important implications for the mechanism of biosynthesis in the TGF-ß family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-ß family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved and which are perturbed by crystal lattice contacts.

Comparing sequence and structure of falcipains and human homologs at prodomain and catalytic active site for malarial peptide based inhibitor design.

BACKGROUND: Falcipains are major cysteine proteases of Plasmodium falciparum involved in haemoglobin degradation and remain attractive anti-malarial drug targets. Several inhibitors against these proteases have been identified, yet none of them has been approved for malaria treatment. Other Plasmodium species also possess highly homologous proteins to falcipains. For selective therapeutic targeting, identification of sequence and structure differences with homologous human cathepsins is necessary. The substrate processing activity of these proteins is tightly controlled via a prodomain segment occluding the active site which is chopped under low pH conditions exposing the catalytic site. Current work characterizes these proteases to identify residues mediating the prodomain regulatory function for the design of peptide based anti-malarial inhibitors. METHODS: Sequence and structure variations between prodomain regions of plasmodial proteins and human cathepsins were determined using in silico approaches. Additionally, evolutionary clustering of these proteins was evaluated using phylogenetic analysis. High quality partial zymogen protein structures were modelled using homology modelling and residue interaction analysis performed between the prodomain segment and mature domain to identify key interacting residues between these two domains. The resulting information was used to determine short peptide sequences which could mimic the inherent regulatory function of the prodomain regions. Through flexible docking, the binding affinity of proposed peptides on the proteins studied was evaluated. RESULTS: Sequence, evolutionary and motif analyses showed important differences between plasmodial and human proteins. Residue interaction analysis identified important residues crucial for maintaining prodomain integrity across the different proteins as well as the pro-segment responsible for inhibitory mechanism. Binding affinity of suggested peptides was highly dependent on their residue composition and length. CONCLUSIONS: Despite the conserved structural and catalytic mechanism between human cathepsins and plasmodial proteases, current work revealed significant differences between the two protein groups which may provide valuable information for selective anti-malarial inhibitor development. Part of this study aimed to design peptide inhibitors based on endogenous inhibitory portions of protease prodomains as a novel aspect. Even though peptide inhibitors may not be practical solutions to malaria at this stage, the approach followed and results offer a promising means to find new malarial inhibitors.

A novel subtilase inhibitor in plants shows structural and functional similarities to protease propeptides.

The propeptides of subtilisin-like serine proteinases (subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana Sequence similarity and the shared ß-α-ß-ß-α-ß core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of Arabidopsis subtilase SBT4.13 with Kd and Ki values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of Arabidopsis SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13.

Alternative cleavage of the bone morphogenetic protein (BMP), Gbb, produces ligands with distinct developmental functions and receptor preferences.

The family of TGF-ß and bone morphogenetic protein (BMP) signaling proteins has numerous developmental and physiological roles. They are made as proprotein dimers and then cleaved by proprotein convertases to release the C-terminal domain as an active ligand dimer. Multiple proteolytic processing sites in Glass bottom boat (Gbb), the Drosophila BMP7 ortholog, can produce distinct ligand forms. Cleavage at the S1 or atypical S0 site in Gbb produces Gbb15, the conventional small BMP ligand, whereas NS site cleavage produces a larger Gbb38 ligand. We hypothesized that the Gbb prodomain is involved not only in regulating the production of specific ligands but also their signaling output. We found that blocking NS cleavage increased association of the full-length prodomain with Gbb15, resulting in a concomitant decrease in signaling activity. Moreover, NS cleavage was required in vivo for Gbb-Decapentaplegic (Dpp) heterodimer-mediated wing vein patterning but not for Gbb15-Dpp heterodimer activity in cell culture. Gbb NS cleavage was also required for viability through its regulation of pupal ecdysis in a type II receptor Wishful thinking (Wit)-dependent manner. In fact, Gbb38-mediated signaling exhibits a preference for Wit over the other type II receptor Punt. Finally, we discovered that Gbb38 is produced when processing at the S1/S0 site is blocked by O-linked glycosylation in third instar larvae. Our findings demonstrate that BMP prodomain cleavage ensures that the mature ligand is not inhibited by the prodomain. Furthermore, alternative processing of BMP proproteins produces ligands that signal through different receptors and exhibit specific developmental functions.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P).

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease.

Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall.

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

The Role of BDNF in the Development of Fear Learning.

Brain-derived neurotrophic factor (BDNF) is a growth factor that is dynamically expressed in the brain across postnatal development, regulating neuronal differentiation and synaptic plasticity. The neurotrophic hypothesis of psychiatric mood disorders postulates that in the adult brain, decreased BDNF levels leads to altered neural plasticity, contributing to disease. Although BDNF has been established as a key factor regulating the critical period plasticity in the developing visual system, it has recently been shown to also play a role in fear circuitry maturation, which has implications for the emergence of fear-related mood disorders. This review provides a detailed overview of developmental changes in expression of BDNF isoforms, as well as their receptors across postnatal life. In addition, recent developmental studies utilizing a genetic BDNF single nucleotide polymorphism (Val66Met) knock-in mouse highlight the impact of BDNF on fear learning during a sensitive period spanning the transition into adolescent time frame. We hypothesize that BDNF in the developing brain regulates fear circuit plasticity during a sensitive period in early adolescence, and alterations in BDNF expression (genetic or environmental) have a persistent impact on fear behavior and fear-related disorders.

The regulatory mechanism of the caspase 6 pro-domain revealed by crystal structure and biochemical assays.

Caspase 6 (CASP6) is a neuron degeneration-related protease and is widely considered to be a potential drug-design target against neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. The N-terminal pro-peptide of CASP6, also referred to as the pro-domain, contains 23 residues and its functional role remains elusive. In this study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. Although the pro-domain was flexible in the crystal, without visible electron density, structural analyses combined with biochemical assays revealed that the pro-domain inhibited CASP6 auto-activation by inhibiting intramolecular cleavage at the intersubunit cleavage site TEVD(193) and also by preventing this site from intermolecular cleavage at low protein concentration through a so-called `suicide-protection' mechanism. Further experiments showed that the length of the pro-domain and the side chain of Asn18 played critical roles in suicide protection. These results disclosed a new inhibitory mechanism of CASP6 and shed light on the pathogenesis and therapeutically relevant study of CASP6-related neurodegenerative diseases.