Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity.

The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases. The solution structure of the complex between the N-terminal inhibitory domain, DID, and the C-terminal DAD, resolved by NMR spectroscopy shows only transient interactions between DID and the basic motif of DAD, resembling those found in encounter complexes. Cross-linking studies placed the RRKR motif into the negatively charged cavity of DID. Neutralizing the cavity resulted in a 5-fold decrease in the binding affinity and 4-fold decrease in the association rate constant of DAD for DID, indicating that the RRKR interactions with DID form a productive encounter complex. A DIAPH1 mutant containing a neutralized RRKR binding cavity shows excessive colocalization with actin and is unresponsive to RAGE stimulation. This is the first demonstration of a specific alteration of the surfaces responsible for productive encounter complexation with implications for human pathology.

Ribosome-Amplified Metabolism, RAMBO, Measured by NMR Spectroscopy.

NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities. NMR spectroscopy and chemical cross-linking combined with high-resolution mass spectrometry were used to establish that PK binds to ribosome at three independent sites, the L1 stalk, the A site, and the mRNA entry pore. The bioanalytical methodology described characterizes the altered kinetics and confirms the specificity of pyruvate kinase-ribosome interaction, affording an opportunity to investigate the ribosome dependence of metabolic reactions under solution conditions that closely mimic the cytosol. Expanding on the concept of ribosomal heterogeneity, which describes variations in ribosomal constituents that contribute to the specificity of cellular processes, this work firmly establishes the reciprocal process by which ribosome-dependent quinary interactions affect metabolic activity.

Effect of a single intravitreal bevacizumab injection on contrast sensitivity and macular thickness in eyes with macular edema from central retinal vein occlusion: a prospective, nonrandomized, three-month follow-up study.

PURPOSE: To evaluate the results of intravitreal bevacizumab (IVB) injection on contrast sensitivity (CS), best-corrected visual acuity (BCVA), foveal thickness (FT) and macular volume (MV) as measured by optical coherence tomography in patients with macular edema (ME) from central retinal vein occlusion (CRVO). METHODS: Sixteen consecutive eyes from 16 patients with ME from unilateral CRVO were treated with a single IVB injection. The CS, BCVA, FT and MV measurements were obtained before the treatment and 1 and 3 months after the injection. RESULTS: CS demonstrated significant improvement at all spatial frequencies - 1.5, 3, 6, 12 and 18 cycles per degree (cpd) - 1 month after the injection and at 6 cpd at the 3-month follow-up. The mean BCVA measurements in log of the minimum angle of resolution (logMAR) units improved from 1.03 at baseline to 0.83 logMAR 1 month after the injection, but worsened to 0.97 logMAR at 3 months. The mean baseline FT ± standard deviation (SD; 620.06 ± 177.60 µm) was reduced significantly 1 month (270.93 ± 74.17 µm) and 3 months (535.56 ± 222.33 µm) after the treatment. The mean baseline MV ± SD (12,765.56 ± 3,769.70 mm(3)) was reduced significantly at the 1-month (8,324.93 ± 932.04 mm(3)) and 3-month (11,319.44 ± 3,044.74 mm(3)) follow-up visits. CONCLUSIONS: IVB improved CS, BCVA, FT and MV within a short time period (1 month). Although VA was not improved at 3 months, improvements were observed for CS, FT and MV, which indicates that, despite ME recurrence, there still was some benefit to visual function.

Ribosome External Electric Field Regulates Metabolic Enzyme Activity: The RAMBO Effect.

Ribosomes bind to many metabolic enzymes and change their activity. A general mechanism for ribosome-mediated amplification of metabolic enzyme activity, RAMBO, was formulated and elucidated for the glycolytic enzyme triosephosphate isomerase, TPI. The RAMBO effect results from a ribosome-dependent electric field-substrate dipole interaction energy that can increase or decrease the ground state of the reactant and product to regulate catalytic rates. NMR spectroscopy was used to determine the interaction surface of TPI binding to ribosomes and to measure the corresponding kinetic rates in the absence and presence of intact ribosome particles. Chemical cross-linking and mass spectrometry revealed potential ribosomal protein binding partners of TPI. Structural results and related changes in TPI energetics and activity show that the interaction between TPI and ribosomal protein L11 mediate the RAMBO effect.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress.

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.

Hydrophobic residues of melittin mediate its binding to αA-crystallin.

The molecular chaperone αA-crystallin, mainly localized in the human ocular lens, is believed to protect the lens from opacification and cataract, by suppressing the aggregation of the other lens proteins. The present study provides structural and thermodynamic insights into the ability of human αA-crystallin (HAA) to bind to its partially unfolded clients in the lens, using a small peptide, melittin from bee venom, as a model client. We characterized the thermodynamic parameters of the binding process between melittin and HAA through isothermal titration calorimetry (ITC), and found the binding to be endothermic and entropy-driven. We identified the amino acids in melittin important for binding to HAA by saturation-transfer difference (STD) nuclear magnetic resonance (NMR) experiments, and analysis of NMR line broadening upon titration of melittin with HAA. Our results suggest that hydrophobic residues Ile17 and Ile20 on the C-terminal region of melittin are in close contact with HAA in the melittin-HAA complex. Information obtained from NMR experiments was used to generate structural models of the melittin-HAA complex by molecular docking with high-ambiguity driven docking (HADDOCK). Structural models of the melittin-HAA complex reveal important principles underlying the interaction of HAA with its clients.

The effects of low-level Pb on steroidogenic acute regulatory protein (StAR) in the prepubertal rat ovary.

Estradiol (E2) is suppressed in prepubertal females exposed maternally to lead (Pb); thus, we assessed effects of Pb on ovarian steroidogenic acute regulatory protein (StAR) as a potential mechanism for this action. Adult Fisher 344 females were dosed with 12 mg of lead acetate per ml of Pb acetate (PbAc) or sodium acetate (NaAc; control), beginning 30 days prior to breeding and continuing until their pups were weaned. For the first part of this study, animals from both groups were killed when 31 days old, at 0800 h, for assessment of basal ovarian StAR gene expression. Results indicated Pb decreased (p < 0.01) both StAR transcripts. In the second part of the study, pregnant mare serum gonadotropin (PMSG) was administered to half of the Pb-treated and control animals at 0800 h. These animals, and animals from both groups that did not receive PMSG, were killed and ovaries and blood collected at 1600 h to assess ovarian StAR protein and E2 responsiveness to gonadotropin stimulation. Pb decreased (p < 0.0001) basal StAR protein expression and lowered (p < 0.001) E2 levels in animals that did not receive PMSG. PMSG induced (p < 0.0001) StAR protein in both the Pb-treated and control animals, an action associated with increased (p < 0.001) serum levels of E2. These results are the first to show that Pb alters basal StAR synthesis, but does not alter gonadotropin-stimulated StAR synthesis, hence, suggesting the primary action of Pb to suppress E2 is through its known action to suppress the serum levels of luteinizing hormone and not due to decreased responsiveness of StAR synthesizing machinery.