Ligand-bound Complexes Research Articles

Blocking Protein kinase C signaling pathway: mechanistic insights into the anti-leishmanial activity of prospective herbal drugs from Withania somnifera

BackgroundLeishmaniasis is caused by several species of leishmania protozoan and is one of the major vector-born diseases after malaria and sleeping sickness. Toxicity of available drugs and drug resistance development by protozoa in recent years has made Leishmaniasis cure difficult and challenging. This urges the need to discover new antileishmanial-drug targets and antileishmanial-drug development.ResultsTertiary structure of leishmanial protein kinase C was predicted and found stable with a RMSD of 5.8Å during MD simulations. Natural compound withaferin A inhibited the predicted protein at its active site with -28.47 kcal/mol binding free energy. Withanone was also found to inhibit LPKC with good binding affinity of -22.57 kcal/mol. Both withaferin A and withanone were found stable within the binding pocket of predicted protein when MD simulations of ligand-bound protein complexes were carried out to examine the consistency of interactions between the two.ConclusionsLeishmanial protein kinase C (LPKC) has been identified as a potential target to develop drugs against Leishmaniasis. We modelled and refined the tertiary structure of LPKC using computational methods such as homology modelling and molecular dynamics simulations. This structure of LPKC was used to reveal mode of inhibition of two previous experimentally reported natural compounds from Withania somnifera - withaferin A and withanone.

MS‐based ligand binding assays with speed, sensitivity, and specificity

Immunoassays are widely used in biochemical/clinical laboratories owing to their simplicity, speed, and sensitivity. We combined self-assembled monolayer-based immunoassays with MALDI-TOF MS to show that high-fidelity surface preparations with a novel matrix deposition/crystallization technique permits quantitative analysis of monolayer-bound antigens at picomolar detection limits. Calibration curves for intact proteins are possible over a broad concentration range and improved specificity of MS-immunoassays is highlighted by simultaneous label-free quantitation of ligand-bound protein complexes.

A data-driven model of a modal gated ion channel: The inositol 1,4,5-trisphosphate receptor in insect Sf9 cells

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel is crucial for the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the complicated ligand regulation of this ubiquitous channel, we constructed a simple quantitative continuous-time Markov-chain model from the data. Our model accounts for most experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel activity established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one complex and indicated that three complexes gate in multiple modes. This also restricted the connectivity between channel complexes. Finally, latencies of channel responses to abrupt ligand concentration changes defined a model with specific network topology between 9 closed and 3 open states. The model with 28 parameters can closely reproduce the equilibrium gating statistics for all three gating modes over a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP3R channel gating behaviors and provide insights to both guide future experiment development and improve IP3R channel gating analysis. Maximum likelihood estimates of the model parameters and of the parameters in the De Young–Keizer model yield strong statistical evidence in favor of our model. Our method is simple and easily applicable to the dynamics of other ion channels and molecules.

Second-chance signal transduction explains cooperative flagellar switching.

The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv–vii).

Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for invivo liver tropism and resistance to invitro immune neutralization. Here, the 4.5Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.

The distribution of ligand-binding pockets around protein-protein interfaces suggests a general mechanism for pocket formation

Protein-protein and protein-ligand interactions are ubiquitous in a biological cell. Here, we report a comprehensive study of the distribution of protein-ligand interaction sites, namely ligand-binding pockets, around protein-protein interfaces where protein-protein interactions occur. We inspected a representative set of 1,611 representative protein-protein complexes and identified pockets with a potential for binding small molecule ligands. The majority of these pockets are within a 6Å distance from protein interfaces. Accordingly, in about half of ligand-bound protein-protein complexes, amino acids from both sides of a protein interface are involved in direct contacts with at least one ligand. Statistically, ligands are closer to a protein-protein interface than a random surface patch of the same solvent accessible surface area. Similar results are obtained in an analysis of the ligand distribution around domain-domain interfaces of 1,416 nonredundant, two-domain protein structures. Furthermore, comparable sized pockets as observed in experimental structures are present in artificially generated protein complexes, suggesting that the prominent appearance of pockets around protein interfaces is mainly a structural consequence of protein packing and thus, is an intrinsic geometric feature of protein structure. Nature may take advantage of such a structural feature by selecting and further optimizing for biological function. We propose that packing nearby protein-protein or domain-domain interfaces is a major route to the formation of ligand-binding pockets.

Second-Chance Signal Transduction is a Model for Bacterial Flagellar Switching and Tropomyosin-Based Motility

The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating unit (motor unit) of the stator. To explain the steeply cooperative switching response to ligand, present models propose allosteric interaction between subunits of the rotor but have yet to address the reaction that stimulates a motor unit to reverse directions. An individual motor unit could exist in ground and excited states corresponding to counterclockwise and clockwise rotation, respectively. After a passing ligand-bound switch complex excites a motor unit, the independent decay rate of the excited state determines the probability that a fresh switch complex will reach the dwell site owing to the steady-state rotation of the rotor before the motor unit returns to ground state. Here, we derive an analytical expression, based on our muscle model, for the energy coupling between a switch complex and a motor unit in the stator complex of a flagellum, and demonstrate that it accounts for the cooperative switching response without the need for allostery. This analytic function becomes the Hill equation as a special case. We found that the analytical result can be reproduced by simulation if the motion of the rotor provides a motor unit with a second chance to remain excited and the outputs from multiple independent motor units are constrained to a single all-or-none event. A motor unit and switch complex represent switch and reader components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and regulated by stochastic ligand binding. By homology, tropomyosin and myosin are modeled in a second chance process as reader and switch respectively. This work was supported by NSF grant MCB-0508203.

A Data Driven Approach to Learning the Kinetics of Single Molecules: A Case Study of Single Inositol 1,4,5-Trisphosphate Receptor Channel

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel plays a central role in the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the crucial intricate ligand regulation of this ubiquitous channel, we constructed the simplest quantitative continuous-time Markov-chain model using a transparent iterative approach, which can be adopted to Markov chain models in general. Our model accounts for all experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel open probability (PO) established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one additional complex and indicated that three of the complexes can gate in two different modes. This also restricted the connectivity between channel complexes. Finally, channel responses to abrupt ligand concentration changes defined a model with 9 closed states and 3 open states, and its network topology. The model with 24 parameters can closely reproduce the equilibrium PO and channel gating statistics for all three gating modes for a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP3R channel gating behaviors not yet explored, and provide insights to both guide future experiment development and improve IP3R channel gating analysis.

Specificity, Structure and Dynamics of Tiam1 PDZ Domain Ligand-Bound Complexes

PSD-95/DlgA/ZO-1 (PDZ) domains are among the most abundant protein-protein interaction domains in the human proteome and typically bind the 4-10 C-terminal residues of its interaction partner with exquisite specificity. We used two homologous PDZ domains from the Tiam-family of guanine nucleotide exchange factors to investigate PDZ specificity. The Tiam1 and Tiam2 PDZ domains have overlapping but distinct ligand binding specificity, and this is exemplified by their unique preferences for C-terminal peptides derived from the syndecan1, Caspr4 and neurexin1 adhesion proteins. The Tiam1 PDZ domain binds syndecan1 and Caspr4 but not neurexin1, while the Tiam2 PDZ domain binds Caspr4 and neurexin1 but not syndecan1. Amino acid sequence comparison of Tiam-family PDZ domains revealed that four residues critical for ligand specificity are not conserved. Remarkably, substitution of these four residues in the Tiam1 PDZ for those found in the Tiam2 PDZ domain switched ligand specificity. To understand the structural and dynamic basis for this change in specificity we used X-ray crystallography and solution NMR methods, respectively. We determined the crystal structures of wild type Tiam1 PDZ domain bound to syndecan1 and phosphorylated syndecan1 peptides and the Tiam1 PDZ quadruple mutant (QM) bound to Caspr4 and neurexin1 peptides. Comparison of the crystal structures of the Tiam1 PDZ-syndecan1 and PDZ-phosphorylated syndecan1 showed that a distinct specificity pocket is used to accommodate the phosphoryl group. The crystal structure of the Tiam1 QM PDZ domain showed a unique side chain stacking interaction between aromatic residues in the PDZ domain and the Caspr4 ligand. Side chain methyl relaxation experiments revealed distinct patterns of dynamics in the Tiam1 PDZ-syndecan1 and PDZ-Caspr4 complexes. Collectively, the structures and dynamics of physiologically-based PDZ domain complexes are contributing to understanding the origin of PDZ specificity and function of Tiam-family PDZ domains.

Influence of ground-state structure and Mg 2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.

Trapping Small Caffeine in a Large GPCR Pocket

Three new X-ray structures of a thermostabilized A 2A adenosine receptor in complex with different antagonists reported by Dore et al. in this issue have advanced the understanding of molecular recognition for this receptor.

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

Homology modeling and dynamics study of aureusidin synthase—An important enzyme in aurone biosynthesis of snapdragon flower

Aurones, a class of plant flavonoids, provide bright yellow color on some important ornamental flowers, such as cosmos, coreopsis, and snapdragon ( Antirrhinum majus). Recently, it has been elucidated that aureusidin synthase (AUS), a homolog of plant polyphenol oxidase (PPO), plays a key role in the yellow coloration of snapdragon flowers. In addition, it has been shown that AUS is a chalcone-specific PPO specialized for aurone biosynthesis. AUS gene has been successfully demonstrated as an attractive tool to engineer yellow flowers in blue flowers. Despite these biological studies, the structural basis for the specificity of substrate interactions of AUS remains elusive. In this study, we performed homology modeling of AUS using Grenache PPO and Sweet potato catechol oxidase (CO). An AUS-inhibitor was then developed from the initial homology model based on the CO and subsequently validated. We performed a thorough study between AUS and PTU inhibitor by means of interaction energy, which indicated the most important residues in the active site that are highly conserved. Analysis of the molecular dynamics simulations of the apo enzyme and ligand-bound complex showed that complex is relatively stable than apo and the active sites of both systems are flexible. The results from this study provide very helpful information to understand the structure–function relationships of AUS.

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S(p))-adenosine cyclic 3':5'-monophosphorothioate ((S(p))-cAMPS)) and the other a diastereoisomeric antagonist ((R(p))-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91-244)·C-subunit, by using fluorescence spectroscopy and amide H/(2)H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91-244), and the effects of cAMP, (S(p))-cAMPS, and (R(p))-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R(p))-cAMPS on amide exchange of the RIα(91-244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91-244). Surprisingly, (R(p))-cAMPS not only increased the affinity of RIα(91-244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R(p))-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R(p))-cAMPS-induced increase in affinity of RIα(91-244) toward the C-subunit indicates that (R(p))-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.

All-Atom Structural Models for Complexes of Insulin-Like Growth Factors IGF1 and IGF2 with Their Cognate Receptor

Type 1 insulin-like growth factor receptor (IGF1R) is a membrane-spanning glycoprotein of the insulin receptor family that has been implicated in a variety of cancers. The key questions related to molecular mechanisms governing ligand recognition by IGF1R remain unanswered, partly due to the lack of testable structural models of apo or ligand-bound receptor complexes. Using a homology model of the IGF1R ectodomain IGF1RΔβ, we present the first experimentally consistent all-atom structural models of IGF1/IGF1RΔβ and IGF2/IGF1RΔβ complexes. Our explicit-solvent molecular dynamics (MD) simulation of apo-IGF1RΔβ shows that it displays asymmetric flexibility mechanisms that result in one of two binding pockets accessible to growth factors IGF1 and IGF2, as demonstrated via an MD-assisted Monte Carlo docking procedure. Our MD-generated ensemble of structures of apo and IGF1-bound IGF1RΔβ agrees reasonably well with published small-angle X-ray scattering data. We observe simultaneous contacts of each growth factor with sites 1 and 2 of IGF1R, suggesting cross-linking of receptor subunits. Our models provide direct evidence in favor of suggested electrostatic complementarity between the C-domain (IGF1) and the cysteine-rich domain (IGF1R). Our IGF1/IGF1RΔβ model provides structural bases for the observation that a single IGF1 molecule binds to IGF1RΔβ at low concentrations in small-angle X-ray scattering studies. We also suggest new possible structural bases for differences in the affinities of insulin, IGF1, and IGF2 for their noncognate receptors.

Using a homology model of cytochrome P450 2D6 to predict substrate site of metabolism

CYP2D6 is an important enzyme that is involved in first pass metabolism and is responsible for metabolizing ~25% of currently marketed drugs. A homology model of CYP2D6 was built using X-ray structures of ligand-bound CYP2C5 complexes as templates. This homology model was used in docking studies to rationalize and predict the site of metabolism of known CYP2D6 substrates. While the homology model was generally found to be in good agreement with the recently solved apo (ligand-free) X-ray structure of CYP2D6, significant differences between the structures were observed in the B' and F-G helical region. These structural differences are similar to those observed between ligand-free and ligand-bound structures of other CYPs and suggest that these conformational changes result from induced-fit adaptations upon ligand binding. By docking to the homology model using Glide, it was possible to identify the correct site of metabolism for a set of 16 CYP2D6 substrates 85% of the time when the 5 top scoring poses were examined. On the other hand, docking to the apo CYP2D6 X-ray structure led to a loss in accuracy in predicting the sites of metabolism for many of the CYP2D6 substrates considered in this study. These results demonstrate the importance of describing substrate-induced conformational changes that occur upon binding. The best results were obtained using Glide SP with van der Waals scaling set to 0.8 for both the receptor and ligand atoms. A discussion of putative binding modes that explain the distribution of metabolic sites for substrates, as well as a relationship between the number of metabolic sites and substrate size, are also presented. In addition, analysis of these binding modes enabled us to rationalize the typical hydroxylation and O-demethylation reactions catalyzed by CYP2D6 as well as the less common N-dealkylation.

The Role of Medical Structural Genomics in Discovering New Drugs for Infectious Diseases

The Role of Medical Structural Genomics in Discovering New Drugs for Infectious Diseases

Interactions of Actinomycin D with Human Telomeric G-Quadruplex DNA

The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticancer drug discovery. The human telomeric G-quadruplex consists of a single strand repeat of d[AGGG(TTAGGG)(3)] that can fold into higher-order DNA structures. Small molecules that selectively target and stabilize the G-quadruplex structure(s) may serve as potential therapeutic agents and have garnered significant interest in recent years. In the work presented here, the anticancer agent, actinomycin D, is demonstrated to bind to and induce changes in both structure and stability in both the Na(+) and K(+) forms of the G-quadruplex DNA. The binding of actinomycin D to the G-quadruplex DNAs is characterized by intrinsic association constants of approximately 2 x 10(5) M(-1) (strand) and 2:1 molecularity, and are shown to be enthalpically driven with binding enthalpies of approximately -7 kcal/mol. The free Na(+) or K(+) forms of the quadruplex structures differ in melting temperatures by approximately 8 degrees C (60 and 68 degrees C, respectively), whereas both forms, when complexed with actinomycin D are stabilized with melting temperatures of approximately 79 degrees C. The induced CD signals observed for the actinomycin D-G-quadruplex complexes may indicate that the phenoxazone ring of actinomycin D is stacked on the G-tetrad rather than intercalated between adjacent G-tetrads. Complex formation with actinomycin D results in changes to both the Na(+) or K(+) structural isoforms to ligand-bound complexes having similar structural properties and stabilities.

The N-terminal domain of GluR6-subtype glutamate receptor ion channels.

The amino terminal domain of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand bound complexes of LIVBP and G-Protein coupled glutamate receptors, and the dimer assembly has a strikingly different conformation from that found in mGluRs. This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain which are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.

Smad-Dependent MicroRNA Processing

Signaling initiated by binding of ligands of the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) families to their cognate receptors stimulates the activity of Smad proteins (see Connections Map by Wrana and Attisano). In the canonical pathway, receptor Smads (R-Smads) are phosphorylated by the ligand-bound receptor complex and then interact with the common Smad, Smad4, which leads to nuclear translocation of the R-Smad/Smad4 complex and regulation of specific genes. Davis et al . show that a Smad4-independent pathway for TGF-β and BMP signaling is mediated by the interaction of R-Smads with specific primary microRNA (miRNA) transcripts and the miRNA processing complex called DROSHA, which contains the RNase III DROSHA, the DiGeorge syndrome critical region gene 8 (DGCR8), and RNA helicases p68 and p72, and that this interaction allows TGF-β and BMP to stimulate the production of the mature miRNAs, which then repress the expression of specific genes. In vascular smooth muscle cells, BMP or TGF-β signaling leads to the increased abundance of smooth muscle cell markers, such as smooth muscle α-actin (SMA). Davis et al . show that BMP2, 4, or 7 stimulated the abundance of mature miR-21 and miR-199a through a nontranscriptional mechanism in human primary pulmonary artery smooth muscle cells (PASMCs). The abundance of the primary miR-21 transcripts did not increase, only the abundance of the precursor and mature miR-21 increased in response to BMP or TGF-β. Expression of the miR-21 target PDCD4 (encoding programmed cell death 4) was decreased in response to BMP. Forced expression of PDCD4 prevented the induction of smooth muscle cell markers by BMP, and knockdown of PDCD4 with small-interfering RNA (siRNA) prevented BMP from stimulating SMA abundance. Knockdown of both BMP-specific R-Smads, Smad1 and Smad5, which are present in PASMCs, prevented BMP-induced increase in the precursor and mature miR-21 ; however, knockdown of Smad4 had no effect. In vitro assays suggested that Smad1, Smad5, and the TGF-β-specific Smad3 interacted with p68 through an RNA-independent mechanism and that BMP stimulated the coimmunoprecipitation of DROSHA and p68 with Smad1 or 5 from PASMCs. RNA chromatin immunoprecipitation experiments revealed that BMP4 stimulated the association of Smad1, but not Smad2 or Smad3, with primary miR-21 or miR-199a , but not miR-214 , and that TGF-β stimulated the association of Smad2 or Smad3, but not Smad1, with primary miR-21 or miR-199a , but not miR-214 . Thus, the interaction between the Smads and the microRNA precursor is ligand specific and microRNA specific. Nuclear extracts from Cos7 cells treated with BMP4 or TGF-β exhibited enhanced primary miR-21 processing activity. Promotion of microRNA processing may be a mechanism by which TGF-β signaling contributes to oncogenesis. In the Smad4-deficient breast cancer cell line MDA-MB-468, TGF-β stimulation increased precursor and mature miR-21 abundance, and expression of a dominant-negative TGF-β receptor subunit in these cells decreased the abundance of the precursor miR-21 , suggesting that autocrine signaling by TGF-β through the Smad4-independent miRNA processing pathway may contribute to the cancerous phenotype. B. N. Davis, A. C. Hilyard, G. Lagna, A. Hata, SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454 , 56-61 (2008). [PubMed]