The effects of pK(a) tuning on the thermodynamics and kinetics of folding: design of a solvent-shielded carboxylate pair at the a-position of a coiled-coil

The tuning of the pK_a of ionizable residues plays a critical role in various protein functions, such as ligand-binding, catalysis, and allostery. Proteins harness the free energy of folding to position ionizable groups in highly specific environments that strongly affect their pK_a values. To investigate the interplay among protein folding kinetics, thermodynamics, and pK_a modulation, we introduced a pair of Asp residues at neighboring interior positions of a coiled-coil. A single Asp residue was replaced for an Asn side chain at the a-position of the coiled-coil from GCN4, which was also crosslinked at the C-terminus via a flexible disulfide bond. The thermodynamic and kinetic stability of the system was measured by circular dichroism and stopped-flow fluorescence as a function of pH and concentration of guanidine HCl. Both sets of data are consistent with a two-state equilibrium between fully folded and unfolded forms. Distinct pK_a values of 6.3 and 5.35 are assigned to the first and second protonation of the Asp pair; together they represent an energetic difference of 5 kcal/mol relative to the protonation of two Asp residues with unperturbed pK_a values. Analysis of the rate data as a function of pH and denaturant concentration allowed calculation of the kinetic constants for the conformational transitions of the peptide with the Asp residues in the doubly protonated, singly protonated, and unprotonated forms. The doubly and singly protonated forms fold rapidly, and a ϕ-value analysis shows that their contribution to folding occurs subsequent to the transition state ensemble for folding. By contrast, the doubly charged state shows a reduced rate of folding and a ϕ-value near 0.5 indicative of a repulsive interaction, and possibly also heterogeneity in the transition state ensemble.     

Cleavage of von Willebrand factor by granzyme M destroys its factor VIII binding capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.     

Mapping parathyroid hormone, β-globin,insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes

Summary Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, -globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, -globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.     

Sensory,motor, and postganglionic sympathetic neurons forming the ulnar and radial nerves of two macaque species: Macaca nemestrina and Macaca fascicularis

The motor, sensory, and postganglionic sympathetic neurons forming the left ulnar and right radial nerves of long-tailed macaques (Macaca fascicularis) and pigtailed macaques (Macaca nemestrina) were localized by the horseradish peroxidase method of tracing neuronal connections. The ulnar and radial motoneurons formed a longitudinal column of variable extent in the lateral part of the ventral horn. In most animals, the ulnar motoneurons extended between the caudal ends of the C7 and T1 segments; the radial motoneurons extended between the rostral level of the C4 and the middle part of T1 segments. Although there were areas of overlap in the spinal distribution of ulnar and radial motoneurons, the ulnar motoneurons were located more dorsally and dorsolaterally than were the radial motoneurons. In most animals, labelled sensory neurons whose axons run with the ulnar nerve occurred in the C8–T4 dorsal root ganglia, and those whose axons run with the radial nerve occurred in the C5–T3 ganglia. The radial sympathetic neurons were distributed in stellate through T7 paravertebral sympathetic ganglia, and the ulnar sympathetic neurons were distributed in stellate through T4 paravertebral sympathetic ganglia. Though the motor, sensory, and sympathetic neurons forming the ulnar and radial nerves had wide segmental distributions, all showed peak frequencies in two segments. The cross-sectional areas of the motor, sensory, and postganglionic sympathetic neurons forming the radial and ulnar nerves were measured in the animal that showed the greatest amount of labelling for each nerve. The ulnar and radial motoneurons had a similar range of sizes, with cross-sectional areas between 120 and 2,160 μm². Most were smaller than 900 μm². The sensory neurons forming the ulnar and radial nerves also displayed a similar range of sizes, measuring between 120 and 3,360 μm² in cross-sectional area. Most neurons measured between 201 and 800 μm². The ulnar sympathetic neurons measured between 120 and 840 μm², and the radial neurons between 120 and 2,120 μm². In both cases, most neurons measured between 120 and 600 μm². The mean cross-sectional area for the radial sympathetic neurons was, however, larger than that for the ulnar sympathetic neurons.     

Diagnosis of abdominal masses with percutaneous biopsy guided by ultrasound.

OBJECTIVE--To assess the accuracy and safety of percutaneous biopsy of abdominal masses guided by ultrasound. DESIGN--Prospective study. SETTING--Combined gastroenterology service, Scarborough Hospital. PATIENTS--108 Consecutive patients identified as having a discrete mass on diagnostic ultrasound examination of the abdomen. INTERVENTION--A sample of tissue was obtained with an aseptic technique under local anaesthesia: an 18 steel wire gauge needle (Tru-Cut) was mounted in a spring loaded firing device (Biopty gun) that was advanced under simultaneous ultrasound scanning, permitting precise localisation of the target organ. MAIN OUTCOME MEASURE--Results of histological examination of tissue specimens. RESULTS--Biopsy failed in four patients. Adequate histological specimens were obtained in 104 patients with masses in the liver (31), pancreas (37), kidney (10), and adrenal glands (six) and in 20 undiagnosed abdominal and retroperitoneal masses. Follow up was until death or confirmation of the diagnosis. Three complications but no deaths occurred. Malignancy was suspected in 84 patients before biopsy. This was confirmed in 70 patients, in 26 of whom confirmation of dissemination obviated the need for further investigation. In 10 patients biopsy indicated a previously unsuspected primary tumour, and in 12 it showed only a benign lesion. Among 24 patients considered to have benign disease biopsy showed an unsuspected neoplasm in seven. Use of biopsy thus had a major effect on clinical management in 55 patients. Four false negative but no false positive diagnoses resulted from the procedure. CONCLUSION--Percutaneous biopsy of abdominal and retroperitoneal masses under ultrasound guidance is a safe and accurate method of obtaining a histological diagnosis. The results obtained have a considerable effect on clinical management.     

Human Thromboxane A2 Receptor Genetic Variants: In Silico,In Vitro and “In Platelet” Analysis

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C⁶⁸S, V⁸⁰E, E⁹⁴V, A¹⁶⁰T, V¹⁷⁶E, and V²¹⁷I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C⁶⁸S) to normal (V²¹⁷I), with most variants demonstrating functional alteration in binding, expression or activation (V⁸⁰E, E⁹⁴V, A¹⁶⁰T, and V¹⁷⁶E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and “in platelet” evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.