Elucidating the complex membrane binding of a protein with multiple anchoring domains using extHMMM.

Membrane binding is a crucial mechanism for many proteins, but understanding the specific interactions between proteins and membranes remains a challenging endeavor. Coagulation factor Va (FVa) is a large protein whose membrane interactions are complicated due to the presence of multiple anchoring domains that individually can bind to lipid membranes. Using molecular dynamics simulations, we investigate the membrane binding of FVa and identify the key mechanisms that govern its interaction with membranes. Our results reveal that FVa can either adopt an upright or a tilted molecular orientation upon membrane binding. We further find that the domain organization of FVa deviates (sometimes significantly) from its crystallographic reference structure, and that the molecular orientation of the protein matches with domain reorganization to align the C2 domain toward its favored membrane-normal orientation. We identify specific amino acid residues that exhibit contact preference with phosphatidylserine lipids over phosphatidylcholine lipids, and we observe that mostly electrostatic effects contribute to this preference. The observed lipid-binding process and characteristics, specific to FVa or common among other membrane proteins, in concert with domain reorganization and molecular tilt, elucidate the complex membrane binding dynamics of FVa and provide important insights into the molecular mechanisms of protein-membrane interactions. An updated version of the HMMM model, termed extHMMM, is successfully employed for efficiently observing membrane bindings of systems containing the whole FVa molecule.

Membrane Interaction of the Factor VIIIa Discoidin Domains in Atomistic Detail.

A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor VIIIa (FVIIIa), the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly was positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla) domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent nonspecificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2.

Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V.

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV-C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2-25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1-3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed "open" and "closed" states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both "open" and "closed" states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1-3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

Cd-content and temperature dependences of hyperfine fields in CdxFe3-xO4.

Cd-content and temperature dependences of hyperfine fields in CdxFe3-xO4 (0 ≤ x ≤ 0.5) were investigated by means of time-differential perturbed angular correlation spectroscopy with the 111Cd(←111In) probe. It was found that Cd2+ ions selectively occupy the tetrahedral A site in the spinel structure in all the range of the present Cd content x. The magnetic transition temperature TC becomes lower with increasing x due to the interference of the long-range ordering of Fe spins as a result of expansion of the lattice constants by Cd doping. The measurement of room-temperature hyperfine fields at different x shows that the supertransferred magnetic hyperfine field (SMHF) at the probe decreases as x increases in the range of 0 ≤ x ≤ 0.5. Isothermal measurements at 15 K revealed a contrastive phenomenon for the Cd contents up to x = 0.4: the SMHF becomes great with increasing x; however, this increasing trend of the SMHF turns to reduction at x = 0.46. These observations can be explained based on the effect of Cd doping on the antiferromagnetic coupling between Fe ions in the A and B sites.

Possible roles of the AP-1 site in the cytosolic T3 binding protein promoter and insights into its physiological significance.

Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to µ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on µ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in µ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of µ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased µ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of µ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on µ-crystallin mRNA expression.

Accelerating membrane insertion of peripheral proteins with a novel membrane mimetic model.

Characterizing atomic details of membrane binding of peripheral membrane proteins by molecular dynamics (MD) has been significantly hindered by the slow dynamics of membrane reorganization associated with the phenomena. To expedite lateral diffusion of lipid molecules without sacrificing the atomic details of such interactions, we have developed a novel membrane representation, to our knowledge, termed the highly mobile membrane-mimetic (HMMM) model to study binding and insertion of various molecular species into the membrane. The HMMM model takes advantage of an organic solvent layer to represent the hydrophobic core of the membrane and short-tailed phospholipids for the headgroup region. We demonstrate that using these components, bilayer structures are formed spontaneously and rapidly, regardless of the initial position and orientation of the lipids. In the HMMM membrane, lipid molecules exhibit one to two orders of magnitude enhancement in lateral diffusion. At the same time, the membrane atomic density profile of the headgroup region produced by the HMMM model is essentially identical to those obtained for full-membrane models, indicating the faithful representation of the membrane surface by the model. We demonstrate the efficiency of the model in capturing spontaneous binding and insertion of peripheral proteins by using the membrane anchor (γ-carboxyglutamic-acid-rich domain; GLA domain) of human coagulation factor VII as a test model. Achieving full insertion of the GLA domain consistently in 10 independent unbiased simulations within short simulation times clearly indicates the robustness of the HMMM model in capturing membrane association of peripheral proteins very efficiently and reproducibly. The HMMM model will provide significant improvements to the current all-atom models by accelerating lipid dynamics to examine protein-membrane interactions more efficiently.

Molecular determinants of phospholipid synergy in blood clotting.

Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca(2+) in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphingomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-serine-specific and phosphate-specific interactions, respectively.

Gene expression differences in oocytes derived from adult and prepubertal Japanese Black cattle during in vitro maturation.

The present study was carried out to compare the gene expression profiles in oocytes derived from adult and prepubertal Japanese Black cattle during in vitro maturation (IVM) using microarray gene chips (Bovine genome array containing 24,072 probe sets representing over 23,000 transcripts). Microarray experiments were conducted using total RNA isolated from immature [germinal vesicle (GV)] and in vitro matured [metaphase II, (MII)] oocytes derived from adult and prepubertal animals. A total of 333 (1.4%) and 549 (2.3%) genes were differentially expressed between prepubertal vs adult bovine GV and MII stages oocytes, respectively. Of these, 176 and 312 genes were up-regulated, while 157 and 237 were down-regulated in prepubertal when compared with adult GV and MII oocytes, respectively. It was also observed that 695 (2.9%) and 553 (2.3%) genes were differentially expressed between GV vs MII stage oocytes in the adult and prepubertal groups, respectively. Gene ontological classification of the differentially expressed genes revealed that up-regulated genes in adult oocytes were involved in signal transduction, transcriptional control and transport. Quantitative reverse transcription-PCR validated the expression profile of some selected transcripts and confirmed differences in the expression levels of transcripts between adult vs prepubertal groups in both GV and MII stages oocytes as identified by microarray data analysis. This study indicated for the first time that significant number of genes were differentially expressed (>2-fold, p < 0.01) between oocytes derived from adult and those from prepubertal Japanese Black cattle, and this difference increased during IVM.

Repetitive Painting (REPEAT) Irradiation in Stereotactic Radiotherapy Using Helical Tomotherapy.

BACKGROUND/AIM: Stereotactic radiotherapy (SRT) for spine metastases with helical tomotherapy requires a long irradiation time due to the high dose per fraction. Since helical tomotherapy can neither confirm nor correct the position during irradiation, a plan with a long irradiation time cannot be used in actual clinical practice, given the intra-fractional motion error. To address this problem, we devised a method called REPEAT irradiation. PATIENTS AND METHODS: REPEtitive pAinTing (REPEAT) irradiation is a method of dividing the irradiation for a given fraction per day into several sessions and performing the irradiation after position correction using mega-voltage computed tomography images for each session. In order to evaluate how REPEAT irradiation changes irradiation time and the dose-volume histogram (DVH), a planning study with helical tomotherapy was conducted using CT images of a patient with lumbar spine metastasis. RESULTS: In this case, we found that dividing 3 irradiation fractions into 3 sessions per day (i.e., 9 fractions=9 sessions in 3 days) using REPEAT irradiation shortened the irradiation time per session and simultaneously improved dose-volume histogram parameters. CONCLUSION: Although the optimal number of sessions may differ depending on the patient's condition, the fixing method, the irradiation site, and the calculation parameters, REPEAT irradiation does not require any special equipment and is a simple practical treatment method.

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.

Uncovering Membrane-Bound Models of Coagulation Factors by Combined Experimental and Computational Approaches.

In the life sciences, including hemostasis and thrombosis, methods of structural biology have become indispensable tools for shedding light on underlying mechanisms that govern complex biological processes. Advancements of the relatively young field of computational biology have matured to a point where it is increasingly recognized as trustworthy and useful, in part due to their high space-time resolution that is unparalleled by most experimental techniques to date. In concert with biochemical and biophysical approaches, computational studies have therefore proven time and again in recent years to be key assets in building or suggesting structural models for membrane-bound forms of coagulation factors and their supramolecular complexes on membrane surfaces where they are activated. Such endeavors and the proposed models arising from them are of fundamental importance in describing and understanding the molecular basis of hemostasis under both health and disease conditions. We summarize the body of work done in this important area of research to drive forward both experimental and computational studies toward new discoveries and potential future therapeutic strategies.

Time Dependence of Intra-fractional Motion in Spinal Stereotactic Body Radiotherapy.

BACKGROUND/AIM: Positional uncertainty in spinal stereotactic body radiotherapy (SBRT) may cause fatal error, therefore, we investigated the intra-fractional spinal motion during SBRT and its time dependency. PATIENTS AND METHODS: Thirty-one patients who received SBRT using CyberKnife were enrolled in the study. 2D kV X-ray spine images in two directions were taken before and during treatment. Image acquisition intervals during treatment were set at 35-60 sec. Automatic image matchings were performed between the reference digital reconstructed radiography (DRR) and live images, and the spinal position displacements were logged in six translational and rotational directions. If the displacements exceeded 2 mm or 1 degree, the treatment beam delivery was interrupted and the patient position was corrected by moving couch, and the couch adjustments were also logged. Based on the information, the time-dependent accumulated translational and rotational displacements without any couch adjustments were calculated. RESULTS: Spinal position displacements in all translational and rotational directions were correlated with elapsed treatment time. Especially, Right-Left displacements of >1 mm and >2 mm were observed at 4-6 and 8-10 min after treatment initiation, respectively. Rotational displacements in the Yaw direction >1° were observed at 10-15 min after treatment initiation. CONCLUSION: The translational and rotational displacements systematically increased with elapsed treatment time. It is suggested that the spine position should be checked at least every 4-6 min or the treatment time should be limited within 4-6 minutes to ensure the irradiation accuracy within the millimeter or submillimeter range.

Distinct structural and adhesive roles of Ca2+ in membrane binding of blood coagulation factors.

The GLA domain, a common membrane-anchoring domain of several serine protease coagulation factors, is a key element in membrane association and activation of these factors in a highly Ca2+-dependent manner. However, the critical role of Ca2+ ions in binding is only poorly understood. Here, we present the atomic model of a membrane-bound GLA domain by using MD simulations of the GLA domain of human factor VIIa and an anionic lipid bilayer. The binding is furnished through a complete insertion of the omega-loop into the membrane and through direct interactions of structurally bound Ca2+ ions and protein side chains with negative lipids. The model suggests that Ca2+ ions play two distinct roles in the process: the four inner Ca2+ ions are primarily responsible for optimal folding of the GLA domain for membrane insertion, whereas the outer Ca2+ ions anchor the protein to the membrane through direct contacts with lipids.

Blood clotting reactions on nanoscale phospholipid bilayers.

Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in "simple" model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease-cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease-cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes.

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes.

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.

Comparison of sulfation of glycosaminoglycans and glycopeptides from control and virus-transformed baby hamster kidney cells.

Sulfation of glycosaminoglycans and glycopeptides was compared in baby hamster kidney (BHK) cells and the polyoma virus transformants (PY-BHK). Cells were incubated with [3H]glucosamine and [35S]sulfate, and the labeled glycosaminoglycans and glycopeptides were isolated by digesting the cell membrane fraction with pronase followed by gel filtration. Each type of glycosaminoglycan in the void volume fraction (Fraction I) and in the included fraction (Fraction II) was determined by sequential enzymatic digestions. The residue was regarded as being glycopeptides. Of the total 3H radioactivities of glycosaminoglycans in Fraction I from BHK cells, 51% were in dermatan sulfate, 23% were in heparan sulfate, 17% were in chondroitin 4- and 6-sulfates, and 9% were in hyaluronate. PY-BHK cells acquired significantly larger amounts of 3H radioactivities in sulfated glycosaminoglycans than did BHK cells on the basis of cell number. On the basis of protein content, there was no such difference due to higher protein content of PY-BHK cells. The degree of sulfation of the glycosaminoglycans, estimated from the ratio of 35S to 3H and by disaccharide analysis, was significantly lower in PY-BHK cells than in BHK cells. Considerable amounts of 35S radioactivities assigned to sulfated glycopeptides were found in Fraction II from both BHK and PY-BHK cells.

Characterization of a Rana catesbeiana lectin-resistant mutant of leukemia P388 cells.

Sialic acid-binding lectin (SBL-C) from Rana catesbeiana eggs inhibits the growth of tumor cells such as P388 and L1210 leukemia cells (K. Nitta et al., Cancer Res., 54: 920-927, 1994). Here we report the establishment of an SBL-resistant P388 variant cell line, RC-150. Both P388 and RC-150 cells were agglutinated by SBL-C; however, growth of RC-150 cells was unaffected by SBL-C. Cytoplasmic free Ca2+ concentration and transglutaminase activity of RC-150 cells were 0.5 (110 nM) and 3 times (0.62 nmol/mg/min) as high as those of P388 cells, respectively. Microvilli and microplicae were observed on the surface of P388 cells by scanning electron microscopy but were rarely seen on RC-150 cells. Dansylcadaverine-labeled SBL-C bound to both P388 and RC-150 cells. Binding of SBL-C to these tumor cells appears to be mediated by two species of wheat germ agglutinin-stained cell membrane sialoglycoproteins. Labeled SBL-C entered P388 but not RC-150 cells, suggesting that internalized SBL-C acts as an inhibitor of cell proliferation.

Long-term results of the Kumamoto Study on optimal diabetes control in type 2 diabetic patients.

OBJECTIVE: To examine whether intensive glycemic control could decrease the frequency or severity of diabetic microvascular complications, an 8-year prospective study of Japanese patients with type 2 diabetes was performed. RESEARCH DESIGN AND METHODS: A total of 110 patients with type 2 diabetes (55 with no retinopathy [the primary prevention cohort] and 55 with simple retinopathy [the secondary intervention cohort]) were randomly assigned to multiple insulin injection therapy (MIT) groups and administered three or more daily insulin injections or assigned to conventional insulin injection therapy (CIT) groups and administered one or two daily intermediate-acting insulin injections. Worsening of microvascular complications was regularly assessed during 8 years. Two or more steps up in the 19 stages of the modified Early Treatment of Diabetic Retinopathy Study classification in retinopathy and one or more stages up among three stages in nephropathy (normoalbuminuria, microalbuminuria, and albuminuria) were defined as worsening of complications. RESULTS: In both primary prevention and secondary intervention cohorts, the cumulative percentages of worsening in retinopathy and nephropathy were significantly lower (P < 0.05) in the MIT group than in the CIT group. In neurological tests after 8 years, the MIT group showed significant improvement (P < 0.05) in the median nerve conduction velocities (motor and sensory nerves), whereas the CIT group showed significant deterioration (P < 0.05) in the nerve conduction velocities and vibration threshold. From this study, the glycemic threshold to prevent the onset and progression of diabetic microvascular complications was as follows: HbA1c < 6.5%, fasting blood glucose concentration < 110 mg/dl, and 2-h postprandial blood glucose concentration < 180 mg/dl. CONCLUSIONS: Intensive glycemic control can delay the onset and progression of the early stages of diabetic microvascular complications in Japanese patients with type 2 diabetes.