Lian-Wang Guo

Intrinsically disordered gamma-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure.

The retinal phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic αβ-subunits of PDE6 and the α-subunit of transducin (αt). Detailed structural analysis of PDEγ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEγ, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEγ46–87 when bound to αt in the transition-state complex. NMR mapping of the interaction between PDEγ46–87 and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74–87 of PDEγ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to αt, is left free at this stage. These results indicate that the interaction between PDEγ and αt during the phototransduction cascade involves the selection of preconfigured transient conformations.

Juxtaposition of the steroid binding domain-like I and II regions constitutes a ligand binding site in the sigma-1 receptor.

σ-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig σ-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig σ-1 receptor (C94A,V190C), both in a σ-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the σ-1 receptor.

Mechanisms of post-intervention arterial remodelling.

It has been appreciated over the past two decades that arterial remodelling, in addition to intimal hyperplasia, contributes significantly to the degree of restenosis that develops following revascularization procedures. Remodelling appears to be an adventitia-based process that is contributed to by multiple factors including cytokines and growth factors that regulate extracellular matrix or phenotypic transformation of vascular cells including myofibroblasts. In this review, we summarize the currently available information from animal models as well as clinical investigations regarding arterial remodelling. The factors that contribute to this process are presented with an emphasis on potential therapeutic methods to enhance favourable remodelling and prevent restenosis.

The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

The sigma-2 receptor (S2R) is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1) a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG) and haloperidol but not to the selective sigma-1 receptor ligand (+)-pentazocine, and (2) a protein of 18–21 kDa, as shown by specific photolabeling with [3H]-Azido-DTG and [125I]-iodoazido-fenpropimorph ([125I]-IAF). Recently, the progesterone receptor membrane component 1 (PGRMC1), a 25 kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380). To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [3H]-DTG binding to the S2R (Bmax) as well as the DTG-protectable [125I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI = 472 μM and 350 μM, respectively), as determined in competition with [3H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20–80 nM). These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

The Inhibitory γ Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pγ), which interact with the catalytic heterodimer (Pαβ) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pγ/Pαβ interactions play a critical role. The quaternary organization of the αβγγ heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pγ-binding regions on Pα and Pβ through photolabel transfer from various Pγ positions throughout the entire molecule. The Pγ N-terminal Val16–Phe30 region was found to interact with the Pαβ GAFa domain, whereas its C terminus (Phe73–Ile87) interacted with the Pαβ catalytic domain. The interactions of Pγ with these two domains were bridged by its central Ser40–Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pγ and Pαβ. Furthermore, a photocross-linked product αβγ(γ) was specifically generated by the double derivatized Pγ, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pγ molecule binds Pαβ in an extended linear interaction and may even interact with both Pα and Pβ simultaneously.

Asymmetric Interaction between Rod Cyclic GMP Phosphodiesterase γ Subunits and αβ Subunits

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Pαβ) and two identical inhibitory subunits (Pγ). Pαβ is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pγ with Pβ as compared with Pα in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pγ and Pαβ, by differentiating Pγ interaction with each individual Pαβ subunit through radiolabel transfer from various positions throughout the entire Pγ molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pγ makes a major contribution to the interaction with Pαβ. In addition, a striking asymmetry of interaction between the Pγ polycationic region and the Pαβ subunits was observed when the stoichiometry of Pγ versus the Pαβ dimer was below 2. Preferential photolabeling on Pβ from Pγ position 40 and on Pα from position 30 increased while lowering the Pγ/Pαβ ratio, but diminished when the Pγ/Pαβ ratio was over 2. Our finding leads to the conclusion that two classes of Pγ binding sites exist on Pαβ, each composed of GAF domains in both Pα and Pβ, differing from the conventional models suggesting that each Pγ binds only one of the Pαβ catalytic subunits. This new model leads to insight into how the unique Pαβ heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pγ.

Role of the Sigma-1 receptor in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease affecting spinal cord motoneurons (MN) with an associative connection to Frontotemporal Lobar Dementia (FTLD). The endoplasmic reticulum (ER) bound Sigma-1 Receptor (S1R) chaperone protein localizes to specialized ER cisternae within 10 nm of the plasma membrane in spinal cord ventral horn cholinergic post synaptic C-terminals. Removal of the S1R gene in the Superoxide Dismutase-1 (SOD-1) mouse model of ALS exacerbated the neurodegenerative condition and resulted in a significantly reduced longevity when compared to the SOD-1/S1R wild type (WT) mouse. The proposed amelioration of the ALS phenotype by the S1R is likely due to a “brake” on excitation of the MN as evidenced by a reduction in action potential generation in the MN of the WT when compared to the S1R KO mouse MN. Although the precise signal transduction pathway(s) regulated by the S1R in the MN has/have not been elucidated at present, it is likely that direct or indirect functional interactions occur between the S1R in the ER cisternae with voltage gated potassium channels and/or with muscarinic M2 receptor signaling in the post synaptic plasma membrane. Possible mechanisms for regulation of MN excitability by S1R are discussed.

Subcellular Localization of the Sigma-1 Receptor in Retinal Neurons — an Electron Microscopy Study

The Sigma-1 receptor (S1R) is known to play a protective role in the central nervous system including the retina. A major barrier for understanding the underlying mechanism is an ambiguity of S1R subcellular localizations. We thus conducted the first electron microscopy (EM) study of S1R subcellular distribution in the mouse retina. Immuno-EM imaging showed previously under-appreciated S1R presence in photoreceptor cells. Unlike in other cell types in previous reports, in photoreceptor cells S1R was found in the nuclear envelope but not localized in the endoplasmic reticulum (ER), raising a possibility of S1R-mediated modulatory mechanisms different than conventionally thought. While in bipolar cells S1R was detected only in the nuclear envelope, in ganglion cells S1R was identified predominantly in the nuclear envelope and found in the ER as well. A predominant localization of S1R in the nuclear envelope in all three retinal neurons implicates a potential role of S1R in modulating nuclear activities. Moreover, its absence in the plasma membrane and presence in the subsurface ER cisternae that are juxtaposed to the plasma membrane in ganglion cells may lend mechanistic insights generally important for frequently reported S1R modulations of ion channels in neurons.

Periadventitial application of rapamycin-loaded nanoparticles produces sustained inhibition of vascular restenosis.

Open vascular reconstructions frequently fail due to the development of recurrent disease or intimal hyperplasia (IH). This paper reports a novel drug delivery method using a rapamycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs)/pluronic gel system that can be applied periadventitially around the carotid artery immediately following the open surgery. In vitro studies revealed that rapamycin dispersed in pluronic gel was rapidly released over 3 days whereas release of rapamycin from rapamycin-loaded PLGA NPs embedded in pluronic gel was more gradual over 4 weeks. In cultured rat vascular smooth muscle cells (SMCs), rapamycin-loaded NPs produced durable (14 days versus 3 days for free rapamycin) inhibition of phosphorylation of S6 kinase (S6K1), a downstream target in the mTOR pathway. In a rat balloon injury model, periadventitial delivery of rapamycin-loaded NPs produced inhibition of phospho-S6K1 14 days after balloon injury. Immunostaining revealed that rapamycin-loaded NPs reduced SMC proliferation at both 14 and 28 days whereas rapamycin alone suppressed proliferation at day 14 only. Moreover, rapamycin-loaded NPs sustainably suppressed IH for at least 28 days following treatment, whereas rapamycin alone produced suppression on day 14 with rebound of IH by day 28. Since rapamycin, PLGA, and pluronic gel have all been approved by the FDA for other human therapies, this drug delivery method could potentially be translated into human use quickly to prevent failure of open vascular reconstructions.

A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation.

RATIONALE Endovascular interventions performed for atherosclerotic lesions trigger excessive vascular smooth muscle cell (SMC) proliferation leading to intimal hyperplasia. Our previous studies show that following endovascular injury, elevated TGF-β/Smad3 promotes SMC proliferation and intimal hyperplasia. Furthermore in cultured SMCs, elevated TGF-β/Smad3 increases the expression of several Wnt genes. Here we investigate a crosstalk between TGF-β/Smad3 and Wnt/β-catenin signaling and its role in SMC proliferation. METHODS AND RESULTS To mimic TGF-β/Smad3 up-regulation in vivo, rat aortic SMCs were treated with Smad3-expressing adenovirus (AdSmad3) or AdGFP control followed by stimulation with TGF-β1 (or solvent). AdSmad3/TGF-β treatment up-regulated Wnt2b, Wnt4, Wnt5a, Wnt9a, and Wnt11 (confirmed by qRT-PCR and ELISA), and also increased β-catenin protein as detected by Western blotting. Blocking Wnt signaling using a Frizzled receptor inhibitor (Niclosamide) abolished TGF-β/Smad3-induced β-catenin stabilization. Increasing β-catenin through degradation inhibition (using SKL2001) or by adenoviral expression enhanced SMC proliferation. Furthermore, application of recombinant Wnt2b, Wnt4, Wnt5a, or Wnt9a, but not Wnt11, stabilized β-catenin and stimulated SMC proliferation as well. In addition, increased β-catenin was found in the neointima of injured rat carotid artery where TGF-β and Smad3 are known to be up-regulated. CONCLUSIONS These results suggest a novel mechanism whereby elevated TGF-β/Smad3 stimulates the secretion of canonical Wnts which in turn enhances SMC proliferation through β-catenin stabilization. This crosstalk between TGF-β/Smad3 and Wnt/β-catenin canonical pathways provides new insights into the pathophysiology of vascular SMCs linked to intimal hyperplasia.