Constitutive activation of guanylate cyclase by the G86R GCAP1 variant is due to "locking" cation-\u3c0 interactions that impair the activator-to-inhibitor structural transition

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-\u3c0 interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished "phase II" in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-\u3c0 interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions

Molecular recognition of rhodopsin kinase GRK1 and recoverin is tuned by switching intra- and intermolecular electrostatic interactions

G protein-coupled receptor kinase 1 (GRK1) or rhodopsin kinase is under specific control of the neuronal Ca2+-sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation-\u3c0 interaction pair in the recoverin-GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca2+-dependent membrane binding, and protein-protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent binding of recoverin to GRK1, reversing the charge from K to E led to more distortions in the interaction process, but both mutations increased the stability of the protein conformation. Molecular dynamics simulations provided an explanation for these findings as they let us suggest that residue 192 per se is not a major stabilizer of the interaction between recoverin and its target but rather that the native K is involved in a network of switching electrostatic interactions in wild-type recoverin

Neuronal calcium sensor GCAP1 encoded by GUCA1A exhibits heterogeneous functional properties in two cases of Retinitis Pigmentosa

Genetic heterogeneity leading to retinal disorders impairs biological processes by causing, for example, severe disorder of signal transduction in photoreceptor outer segments. A normal balance of the second messenger homeostasis in photoreceptor cells seems to be a crucial factor for healthy and normal photoreceptor function. Genes like GUCY2D coding for guanylate cyclase GC-E and GUCA1A coding for the Ca2+-sensor guanylate cyclase-activating protein GCAP1 are critical for a precisely controlled synthesis of the second messenger cGMP. Mutations in GUCA1A frequently correlate in patients with cone dystrophy and cone-rod dystrophy. Here, we report two mutations in the GUCA1A gene that were found in patients diagnosed with retinitis pigmentosa, a phenotype that was rarely detected among previous cases of GUCA1A related retinopathies. One patient was heterozygous for the missense variant c.55C > T (p.H19Y), while the other patient was heterozygous for the missense variant c.479T > G (p.V160G). Using heterologous expression and cell culture systems, we examined the functional and molecular consequences of these point mutations. Both variants showed a dysregulation of guanylate cyclase activity, either a profound shift in Ca2+-sensitivity (H19Y) or a nearly complete loss of activating potency (V160G). Functional heterogeneity became also apparent in Ca2+/Mg2+-binding properties and protein conformational dynamics. A faster progression of retinal dystrophy in the patient carrying the V160G mutation seems to correlate with the more severe impairment of this variant

Does ultrasonographic volume of the thyroid gland correlate with difficult intubation? An observational study

BACKGROUND AND OBJECTIVES: Preoperative ultrasonographic evaluation of the thyroid gland done by surgeons could let us foresee airway management challenges. The aim of this observational study was to evaluate the effects of thyroid-related parameters assessed preoperatively by surgeons via ultrasonography and chest X-ray on intubation conditions. METHODS: Fifty patients undergoing thyroid surgery were enrolled. Thyromental distance, Mallampati score, neck circumference and range of neck movement were evaluated before the operation. Thyroid volume, signs of invasion or compression and tracheal deviation on chest X-ray were also noted. The intubation conditions were assessed with Cormack and Lehane score and the intubation difficulty scale. Statistical analyses were done with SPSS 15.0 software. RESULTS: The mean thyroid volume of the patients was 26.38 &#177; 14 mL. The median intubation difficulty scale was 1 (0-2). Thyromental distance (p = 0.011; r = 0.36; 95% CI 0.582-0.088), Mallampati score (p = 0.041; r = 0.29; 95% CI 0.013-0.526), compression or invasion signs (p = 0.041; r = 0.28; 95% CI 0.006-0.521) and tracheal deviation on chest X-ray (p = 0.041; r = 0.52; 95% CI 0.268-0.702) were correlated with intubation difficulty scale. Also patients were classified into two groups related to their intubation difficulty scale (Group I, n = 19: intubation difficulty scale = 0; Group II, n = 31: 1 < intubation difficulty scale &#8804; 5) and difficult intubation predictors and thyroid-related parameters were compared. Only Mallampati score was significantly different between groups (p = 0.025). CONCLUSION: The thyroid volume is not associated with difficult intubation. However clinical assessment parameters may predict difficult intubation

Increased expression levels of Syntaxin 1A and Synaptobrevin 2/Vesicle-Associated Membrane Protein-2 are associated with the progression of bladder cancer

Abstract Gene expression is tightly regulated in time and space through a multitude of factors consisting of signaling molecules. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) are membrane proteins responsible for the intercellular trafficking of signals through endocytosis and exocytosis of vesicles. Altered expression of SNARE proteins in cellular communication is the major hallmark of cancer phenotypes as indicated in recent studies. SNAREs play an important role in maintaining cell growth and epithelial membrane permeability of the bladder and are not only involved in cancer progression but also metastatic cell invasion through SNARE-mediated trafficking. Synaptobrevin2/Vesicle associated membrane protein-2 (v-SNARE) and Syntaxin (t-SNARE) form a vesicular docking complex during endocytosis. Some earlier studies have shown a critical role of SNARE in colon, lungs, and breast cancer progression and metastasis. In this study, we analyzed the relative expression of the STX1A and VAMP2 (SYB2) for their possible association in the progression and metastasis of bladder cancer. The profiling of the genes showed a significant increase in STX1A and VAMP2 expression (p < 0.001) in high-grade tumor cells compared to normal and low-grade tumors. These findings suggest that elevated expression of STX1A and VAMP2 might have caused the abnormal progression and invasion of cancer cells leading to the transformation of cells into high-grade tumor in bladder cancer

Does ultrasonographic volume of the thyroid gland correlate with difficult intubation? An observational study

BACKGROUND AND OBJECTIVES: Preoperative ultrasonographic evaluation of the thyroid gland done by surgeons could let us foresee airway management challenges. The aim of this observational study was to evaluate the effects of thyroid-related parameters assessed preoperatively by surgeons via ultrasonography and chest X-ray on intubation conditions. METHODS: Fifty patients undergoing thyroid surgery were enrolled. Thyromental distance, Mallampati score, neck circumference and range of neck movement were evaluated before the operation. Thyroid volume, signs of invasion or compression and tracheal deviation on chest X-ray were also noted. The intubation conditions were assessed with Cormack and Lehane score and the intubation difficulty scale. Statistical analyses were done with SPSS 15.0 software. RESULTS: The mean thyroid volume of the patients was 26.38 ± 14 mL. The median intubation difficulty scale was 1 (0-2). Thyromental distance (p = 0.011; r = 0.36; 95% CI 0.582-0.088), Mallampati score (p = 0.041; r = 0.29; 95% CI 0.013-0.526), compression or invasion signs (p = 0.041; r = 0.28; 95% CI 0.006-0.521) and tracheal deviation on chest X-ray (p = 0.041; r = 0.52; 95% CI 0.268-0.702) were correlated with intubation difficulty scale. Also patients were classified into two groups related to their intubation difficulty scale (Group I, n = 19: intubation difficulty scale = 0; Group II, n = 31: 1 < intubation difficulty scale ≤ 5) and difficult intubation predictors and thyroid-related parameters were compared. Only Mallampati score was significantly different between groups (p = 0.025). CONCLUSION: The thyroid volume is not associated with difficult intubation. However clinical assessment parameters may predict difficult intubation