Structure-function relationships

The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobiotic and endobiotic metabolism. The unique properties of human CAR, such as the high constitutive activity and the complexity of signaling, as well as the lack of functional and predictive cell-based assays to study the properties of the receptor, have hindered the discovery of selective human CAR ligands. Here we report a novel human CAR inverse agonist, 1-[(2-methylbenzofuran-3-yl)methyl]-3-(thiophen-2-ylmethyl) urea (S07662), which suppresses human CAR activity, recruits the corepressor NCoR in cell-based assays, and attenuates the phenytoin- and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO)-induced expression of CYP2B6 mRNA in human primary hepatocytes. The properties of S07662 are also compared with those of known human CAR inverse agonists by using an array of different in vitro and in silico assays. The identified compound S07662 can be used as a chemical tool to study the biological functions of human CAR and also as a starting point for the development of new drugs for various conditions involving the receptor.

Kant's analysis of the concept of natural purpose in the Critique of judgment captured several features of organisms that he argued warranted making them the objects of a special field of study, in need of a special regulative teleological principle. By showing that organisms have to be conceived as self-organizing wholes, epigenetically built according to the idea of a whole that we must presuppose, Kant accounted for three features of organisms conflated in the biological sciences of the period: adaptation, functionality and conservation of forms. Kant's unitary concept of natural purpose was subsequently split in two directions: first by Cuvier's comparative anatomy, that would draw on the idea of adaptative functions as a regulative principle for understanding in reconstituting and classifying organisms; and then by Goethe's and Geoffroy's morphology, a science of the general transformations of living forms. However, such general transformations in nature, objects of an alleged 'archaeology of nature', were thought impossible by Kant in §80 of the Critique of judgment. Goethe made this 'adventure of reason' possible by changing the sense of 'explanation': scientific explanation was shifted from the investigation of the mechanical processes of generation of individual organisms to the unveiling of some ideal transformations of types instantiated by those organisms.

An emerging field of human brain imaging deals with the characterization of the connectome, a comprehensive global description of structural and functional connectivity within the human brain. However, the question of how functional and structural connectivity are related has not been fully answered yet. Here, we used different methods to estimate the connectivity between each voxel of the cerebral cortex based on functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI) data in order to obtain observer-independent functional-structural connectomes of the human brain. Probabilistic fiber-tracking and a novel global fiber-tracking technique were used to measure structural connectivity whereas for functional connectivity, full and partial correlations between each voxel pair's fMRI-timecourses were calculated. For every voxel, two vectors consisting of functional and structural connectivity estimates to all other voxels in the cortex were correlated with each other. In this way, voxels structurally and functionally connected to similar regions within the rest of the brain could be identified. Areas forming parts of the 'default mode network' (DMN) showed the highest agreement of structurefunction connectivity. Bilateral precuneal and inferior parietal regions were found using all applied techniques, whereas the global tracking algorithm additionally revealed bilateral medial prefrontal cortices and early visual areas. There were no significant differences between the results obtained from full and partial correlations. Our data suggests that the DMN is the functional brain network, which uses the most direct structural connections. Thus, the anatomical profile of the brain seems to shape its functional repertoire and the computation of the whole-brain functional-structural connectome appears to be a valuable method to characterize global brain connectivity within and between populations.

Hierarchical organization in biology is usually conceptualized in terms of compositional levels of structurally-defined entities. However, composition in biological systems is inescapably a dynamic affair concerning what a component does and not only what it is. This chapter examines how the dissociability of structure and function in complex evolved systems leads to ambiguities in the placement of things at levels, which makes "levels" a less useful concept. To address this difficulty, I develop a framework in which general levels of organization are restricted to levels decomposed under a specific causal process. In this approach, levels include only components and processes contributing to a focal process. After defining interlevel and intralevel relations in the process-relative framework, I explore a proposed decomposition of the process of developmental evolution. The resulting levels of developmental evolution are contrasted with levels of selection and replicator-interactor hierarchies.

Recent successes of systems biology clarified that biological functionality is multilevel. We point out that this fact makes it necessary to revise popular views about macromolecular functions and distinguish between local, physico-chemical and global, biological functions. Our analysis shows that physico-chemical functions are merely tools of biological functionality. This result sheds new light on the origin of cellular life, indicating that in evolutionary history, assignment of biological functions to cellular ingredients plays a crucial role. In this wider picture, even if aggregation of chance mutations of replicator molecules and spontaneously self-assembled proteins led to the formation of a system identical with a living cell in all physical respects but devoid of biological functions, it would remain an inanimate physical system, a pseudo-cell or a zombie-cell but not a viable cell. In the origin of life scenarios, a fundamental circularity arises, since if cells are the minimal units of life, it is apparent that assignments of cellular functions require the presence of cells and vice versa. Resolution of this dilemma requires distinguishing between physico-chemical and biological symbols as well as between physico-chemical and biological information. Our analysis of the concepts of symbol, rule and code suggests that they all rely implicitly on biological laws or principles. We show that the problem is how to establish physico-chemically arbitrary rules assigning biological functions without the presence of living organisms. We propose a solution to that problem with the help of a generalized action principle and biological harnessing of quantum uncertainties. By our proposal, biology is an autonomous science having its own fundamental principle. The biological principle ought not to be regarded as an emergent phenomenon. It can guide chemical evolution towards the biological one, progressively assigning greater complexity and functionality to macromolecules and systems of macromolecules at all levels of organization. This solution explains some perplexing facts and posits a new context for thinking about the problems of the origin of life and mind.

"Background: Brain structure and dynamics are interdependent through processes such as activity-dependent neuroplasticity. In this study, we aim to theoretically examine this interdependence in a model of spontaneous cortical activity. To this end, we simulate spontaneous brain dynamics on structural connectivity networks, using coupled nonlinear maps. On slow time scales structural connectivity is gradually adjusted towards the resulting functional patterns via an
unsupervised, activity-dependent rewiring rule. The present model has been previously shown to generate cortical-like, modular small-world structural topology from initially random connectivity. We provide further biophysical justification for this model and quantitatively characterize the relationship between structure, function and dynamics that accompanies the ensuing self organization.

Results: We show that coupled chaotic dynamics generate ordered and modular functional patterns, even on a random underlying structural connectivity. Consequently, structural
connectivity becomes more modular as it rewires towards these functional patterns. Functional networks reflect the underlying structural networks on slow time scales, but significantly less so on faster time scales. In spite of ordered functional topology, structural networks remain robustly
interconnected – and therefore small-world – due to the presence of central, inter-modular hub nodes. The noisy dynamics of these hubs enable them to persist despite ongoing rewiring and despite their comparative absence in functional networks.

Conclusion: Our results outline a theoretical mechanism by which brain dynamics may facilitate neuroanatomical self-organization. We find time scale dependent differences between structural and functional networks. These differences are likely to arise from the distinct dynamics of central structural nodes."

The constitutive androstane receptor (CAR; NR1I3) has emerged as one of the main drug- and xenobiotic-sensitive transcriptional regulators. It has a major effect on the expression of several oxidative and conjugative enzymes and transporters, and hence, CAR can contribute to drug/drug interactions. Novel functions for CAR are also emerging: it is able to modulate the metabolic fate of glucose, lipids, and bile acids, and it is also involved in cell-cell communication, regulation of the cell cycle, and chemical carcinogenesis. Here, we will review the recent information available on CAR and its target gene expression, its interactions with partner proteins and mechanisms of action, interindividual and species variation, and current advances in CAR ligand selectivity and methods used in interrogation of its ligands.

Force is transmitted from muscle fiber to bone via several pathways: (1) via the tendons (i.e. myotendinous force transmission), (2) via intermuscular connective tissue to adjacent muscles (i.e. intermuscular myofascial force transmission), (3) via structures other than muscles (i.e. extramuscular myofascial force transmission). In vivo, the position of a muscle relative to adjacent muscles changes due to differences in moment arm between synergists as well as due to the fact that some muscles span only one joint and other muscles more than one joint. The position of a muscle relative to non-muscular structures within a compartment is altered with each change of the length of the muscle.

A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XI11 A-chains (FXIII A), designated as AK513, AA502, AY481, and AK462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity. AK513 and AA502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the K,,, for the glutamine substrate and a 2-fold increase in the K,,, of the primary amine substrate. There was no detectable transglutaminase activity for either thrombin-activated AY481 or AK462. The rate of ammonia release of thrombin-activated AK513 and a 5 0 2 was reduced 6-and 4-fold, respectively, whereas ammonia release was not detected for the AY481 and A462 mutants. The Kact for calcium ions of the AK513 mutant was similar to recombinant FXIIIa, whereas, it was increased by -3-fold for the AA502 mutant. The rate of fibrin y-chain dimer formation for the AK513 and AA502 mutants was reduced by -19-fold. AK462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound. In conclusion, these results documented that the carboxyl-terminal calcium binding domain ( A~p~~-G l u~~~) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.

Si rintraccia la genesi del concetto di opera letteraria e artistica come struttura nel pensiero russo del primo Novecento, con attenzione privilegiata ai Formalisti dei quali si evidenziano molteplici letture filosofiche. Ci si sofferma in particolare sulla fortunata traduzione russa (1911) di Philosophie der Kunst del filosofo tedesco Broder Christiansen, scritto fondamentale per larga parte dell'estetica, della teoria della dell'arte e della letteratura russe del periodo. Di questo saggio si mette a fuoco la nozione di dominante, la quale otterrà ampia diffusione grazie soprattutto a Roman Jakobson. Del pensiero del grande linguista emerge la forte matrice teleologica, che pervade del resto l'idea di opera come intero propria di molti Formalisti. Si conclude con qualche rapido accenno all'immagine del dispositivo, oggetto di recenti discussioni in ambito francese.

The Fanconi Anemia pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the Fanconi Anemia core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex at 3.2 Å. The structure reveals an architecture that is fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal RING domain predicted to facilitate E2 binding. Binding assays demonstrate that the DRWD domain, but not the ELF domain, is responsible for substrate binding.

Aims The maternal gametophytic calyptra is critical for moss sporophyte development and ultimately sporogenesis. The calyptra has been predicted to protect the sporophyte apex, including the undifferentiated sporogenous region and seta meristem, from desiccation. We investigate the hypothesis that this waterproofing ability is due to a waxy cuticle. The idea that moss calyptrae are covered by a cuticle has been present in the literature for over a century, but, until now, neither the presence nor the absence of a cuticle has been documented for any calyptra. † Methods The epidermis of the calyptra, leafy gametophyte and sporophyte sporangia of the moss Funaria hygrometrica were examined using scanning and transmission electron microscopy. Thicknesses of individual cuticle layers were quantified and compared statistically. The immunochemistry antibody (LM19) specific for pectins was used to locate cell wall material within the cuticle. † Key Results A multi-layered cuticle is present on the calyptra of F. hygrometrica, including layers analogous to the cuticular layer, cell wall projections, electron-lucent and electron-dense cuticle proper observed in vascular plants. The calyptra rostrum has a cuticle that is significantly thicker than the other tissues examined and differs by specialized thickenings of the cuticular layer (cuticular pegs) at the regions of the anticlinal cell walls. This is the first documentation of cuticular pegs in a moss. † Conclusions The calyptra and its associated cuticle represent a unique form of maternal care in embryophytes. This organ has the potential to play a critical role in preventing desiccation of immature sporophytes and thereby may have been essential for the evolution of the moss sporophyte.

Linking durability of carbon blacks, expressed as their oxidation resistance, used in PEMFCs as catalyst supports, with their chemistry and morphology is an important task towards designing carbon blacks with desired properties. Structure-to-property relationship between surface chemistry determined by X-ray photoelectron spectroscopy (XPS), morphological structure determined by digital image processing of scanning electron microscopy (SEM) images, physical properties, and electrochemical corrosion behavior determined in an air-breathing gas-diffusion electrode is studied for several un-altered and several modified carbon blacks. We are showing that surface chemistry, graphitic content and certain physical characteristics such as Brunauer–Emmett–Teller (BET) surface area and pore volume, determined by nitrogen adsorptions are not sufficient to explain high corrosion instability of types of carbon blacks. Inclusion of morphological characteristics, such as roughness, texture and shape parameters provide for more inclusive description and therefore more complete structure-to-property correlations of corrosion behavior of carbon blacks. This paper presents the first direct statistically-derived structure-to-property relationship, developed by multivariate analysis (MVA) that links chemical and physical structural properties of the carbon blacks to their critical properties as supports for PEMFC catalysts. We have found that balance between electrocatalytic activity and high resistance towards oxidation and corrosion is achieved by balance between amount of graphitic content and surface oxide coverage, smaller overall roughness and, finally, larger amount of big elongated and loose, and, hypothetically, more hydrophobic pores.

Si rintraccia la genesi del concetto di opera letteraria e artistica come struttura nel pensiero russo del primo Novecento, con attenzione privilegiata ai Formalisti dei quali si evidenziano molteplici letture filosofiche. Ci si sofferma in particolare sulla fortunata traduzione russa (1911) di Philosophie der Kunst del filosofo tedesco Broder Christiansen, scritto fondamentale per larga parte dell'estetica, della teoria della dell'arte e della letteratura russe del periodo. Di questo saggio si mette a fuoco la nozione di dominante, la quale otterrà ampia diffusione grazie soprattutto a Roman Jakobson. Del pensiero del grande linguista emerge la forte matrice teleologica, che pervade del resto l'idea di opera come intero propria di molti Formalisti. Si conclude con qualche rapido accenno all'immagine del dispositivo, oggetto di recenti discussioni in ambito francese.

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endo-somes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5′-(β, γ)-imidotriphos-phate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure , function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.

Tendon exhibits nonlinear stress-strain behavior that may be partly due to movement of collagen fibers through the extracellular matrix. While a few techniques have been developed to evaluate the fiber architecture of other soft tissues, the organizational behavior of tendon under load has not been determined. The supraspinatus tendon (SST) of the rotator cuff is of particular interest for investigation due to its complex mechanical environment and corresponding inhomogeneity. In addition, SST injury occurs frequently with limited success in treatment strategies, illustrating the need for a better understanding of SST properties. Therefore, the objective of this study was to quantitatively evaluate the inhomogeneous tensile mechanical properties, fiber organization, and fiber realignment under load of human SST utilizing a novel polarized light technique. Fiber distributions were found to become more aligned under load, particularly during the low stiffness toe-region, suggesting that fiber realignment may be partly responsible for observed nonlinear behavior. Fiber alignment was found to correlate significantly with mechanical parameters, providing evidence for strong structure-function relationships in tendon. Human SST exhibits complex, inhomogeneous mechanical properties and fiber distributions, perhaps due to its complex loading environment. Surprisingly, histological grade of degeneration did not correlate with mechanical properties. ß

Teeth adopt a variety of different morphologies, each of which is presumably optimized for performing specific functions during feeding. It is generally agreed that the enamel cap is a crucial element in controlling the mechanical behavior of mammalian teeth under load. Incisors are particularly interesting in terms of structure-function relations, as their role in feeding is that of the 'first bite'. However, little is known how incisor cap morphology is related to tooth deformation. In the present paper we examine the mechanical behavior of mandibular central incisors in the cercopithecine primate Macaca mulatta under loads similar to those encountered during ingestion. We map three-dimensional displacements on the labial surface of the crown as it is compressed, using electronic speckle pattern interferometry (ESPI), an optical metrology method. In addition, micro-computed tomography is used to obtain data regarding the morphology of the enamel cap, which in the M. mulatta lower incisors exhibits missing or very little enamel on the lingual face. The results showed that although compressed along a longitudinal axis, deformation in the incisors mostly occurred in the lingual direction and orthogonal to the direction of the applied load. Both isolated, embedded teeth and teeth in the mandible showed considerable lingual deformation. Incisor deformation in the mandible was generally greater, reflecting the additional freedom of movement enabled by the supporting structures. We show that the association with adjacent teeth in the arch is significant for the behavior of the tooth under load. Finally, loading two teeth simultaneously in the mandible showed that they work as one functional unit. We suggest that these results demonstrate the importance of enamel cap morphology in directing deformation behavior; an ability stemming from the stiffness of the enamel cap overlying the more pliable dentin.

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold. While, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a nonhydrolyzable GTP analog guanosine-5’-(β,γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite of an in silico approach, this
study provides a deep insight into the impact of mutation on the structure, function, stability and mechanism of binding of GppNHp to the Rab5a at molecular level.

The subscapularis is an important mover and stabilizer of the glenohumeral joint. Since the advent of shoulder arthroscopy, partial tears are found in 43% of rotator cuff patients. While partial tears to the upper band occur more commonly, little is known about the structure and mechanical behavior of the individual bands. Therefore, the objective of this study was to measure tensile mechanical properties, corresponding collagen fiber alignment, and histology in the upper and lower bands of the rat subscapularis tendon.
MATERIALS AND METHODS:
Thirty adult Sprague-Dawley rats were euthanized and subscapularis tendons dissected out for mechanical organization (n = 24) and histologic assessment (n = 6). Collagen organization was measured with a custom device during mechanical testing.
RESULTS:
Linear-region modulus at the insertion site was significantly lower in the upper band compared to the lower band, while no differences were found at the midsubstance location. The upper band was found to be significantly less aligned and demonstrated a more rounded cell shape than the lower band at the insertion site.
DISCUSSION:
This study demonstrated that the 2 bands of the subscapularis tendon have differential mechanical, organizational, and histological properties, which suggests a functional deficit exists to the upper band of the subscapularis and may be contributing to the prevalence of partial subscapularis tears.
CONCLUSIONS:
Clinicians should be aware that the upper band of the subscapularis tendon may be at higher risk of developing tears, based on decreased mechanical properties and a more disorganized collagen fiber distribution.

Late Embryogenesis Abundant Proteins (LEAPs) are ubiquitous proteins expected to play major roles in desiccation
tolerance. Little is known about their structure - function relationships because of the scarcity of 3-D structures for LEAPs.
The previous building of LEAPdb, a database dedicated to LEAPs from plants and other organisms, led to the classification
of 710 LEAPs into 12 non-overlapping classes with distinct properties. Using this resource, numerous physico-chemical
properties of LEAPs and amino acid usage by LEAPs have been computed and statistically analyzed, revealing distinctive
features for each class. This unprecedented analysis allowed a rigorous characterization of the 12 LEAP classes, which
differed also in multiple structural and physico-chemical features. Although most LEAPs can be predicted as intrinsically
disordered proteins, the analysis indicates that LEAP class 7 (PF03168) and probably LEAP class 11 (PF04927) are natively
folded proteins. This study thus provides a detailed description of the structural properties of this protein family opening
the path toward further LEAP structure - function analysis. Finally, since each LEAP class can be clearly characterized by a
unique set of physico-chemical properties, this will allow development of software to predict proteins as LEAPs.

Experimental data derived from two classes of superior colliculus neurons*deep layer neurons (DLNs) and super"cial layer neurons (SLNs)*have been used to study the in#uence of dendritic anatomy on synaptic input processing. Anatomical measures of dendrites were "rst determined. Compartmental neuron models (3 of each class) were built to estimate passive membrane parameters and to calculate functional characteristics of synaptic input (attenuation, delay and time window). The two cell classes show distinct di!erences both in morphological and functional characteristics. While the functional parameters derived on SLNs are compatible with time-critical functions, DLNs in contrast show integrator traits..

Tissue transglutaminase (tTG) catalyzes a Ca 2؉dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (K m ‫؍‬ 38 ؎ 10 M) at a 3-fold greater steady-state affinity than with Mg-GTP (K m ‫؍‬ 130 ؎ 35 M). In addition, Mg-ATP inhibited GTP hydrolysis (IC 50 ‫؍‬ 24 M), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC 50 values of 9, 9, and 400 M, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

The cellular environment is crowded by macromolecules of various sizes, shapes, and charges, which modulate protein structure, function and dynamics. Herein, we contemplated the effect of three different macromolecular crowders: dextran-40, ficoll-70 and PEG-35 on the structure, active-site conformational dynamics, function and relative domain movement of multi-domain human serum albumin (HSA). All the crowders used in this study have zero charges and similar sizes (at least in the dilute region) but different shapes and compositions. Some observations follow the traditional crowding theory. For example, all the crowders increased the α-helicity of HSA and hindered the conformational fluctuation dynamics. However, some observations are not in line with the expectations, as an increase in the size of HSA with PEG-35 and uncorrelated domain movement of HSA with ficoll-70 and PEG-35. The relative domain movement is correlated with the activity, suggesting that such moves are essential for protein function. The interaction between HSA and ficoll-70 is proposed to be hydrophobic in nature. Overall, our results provide a somewhat systematic study of the shape-dependent macromolecular crowding effect on various protein properties and present a possible new insight into the mechanism of macromolecular crowding

The complex relationship between structural connectivity (SC) and functional connectivity (FC) of human brain networks is still a critical problem in neuroscience. In order to investigate the role of SC in shaping resting-state FC, numerous models have been proposed. Here, we use a simple dynamic model based on the susceptible-infected-susceptible (SIS) model along the shortest paths to predict FC from SC. Unlike the previous dynamic model based on SIS theory, we focus on the shortest paths as the principal routes to transmit signals rather than the empirical structural brain network. We first simplify the structurally connected network into an efficient propagation network according to the shortest paths and then combine SIS infection theory with the efficient network to simulate the dynamic process of human brain activity. Finally, we perform an extensive comparison study between the dynamic models embedded in the efficient network, the dynamic model embedded in the structurally connected network and dynamic mean field (DMF) model predicting FC from SC. Extensive experiments on two different resolution datasets indicate that ⅰ) the dynamic model simulated on the shortest paths can predict FC among both structurally connected and unconnected node pairs; ⅱ) though there are fewer links in the efficient propagation network, the predictive power of FC derived from the efficient propagation network is better than the dynamic model simulated on a structural brain network; ⅲ) in comparison with the DMF model, the dynamic model embedded in the shortest paths is found to perform better to predict FC.

Lipases are of immense importance in insects since lipids constitute a major dietary component of insects such as T. castaneum. We performed computational analysis for exploring structural features including catalytic properties of a neutral lipase from T. castaneum. The protein shared significant identity with human pancreatic lipase (HPL) and other mammalian pancreatic lipases and related proteins. Two major domains were identified: N-terminal domain and C-terminal domain, connected by a hinge region. N-terminal domain had the following features, similar to pancreatic lipases: signal peptide, α/β hydrolase fold, catalytic triad, nucleophilic elbow, oxyanion hole, lid domain, β5 loop, β9 loop, three N-glycosylation sites, and four disulfide bonds. Lid domain covers active site and renders it inaccessible to the solvent; displacement of lid should expose the active site for substrate binding by interfacial activation. Arrangement of the catalytic triad, lid domain, β5 loop and β9 loops of T. castaneum neutral lipase and a previously reported T. castaneum acidic lipase were compared to highlight the differences. More experimental data is needed to ascertain the function and mechanism of T. castaneum neutral lipase.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein “core complex” containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipi-colinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named " bislysine " , resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a K i near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development.

Mixing generated by gravitational acceleration and the role of local turbulence measured through multifractal methods is examined in numerical experiments of Rayleigh-Taylor and Richtmyer-Meshkov driven front occurring at density interfaces. The global advance of the fronts is compared with laboratory experiments and Nusselt and Sherwood numbers are calculated in both Large Eddy Simulation (LES) and Kinematic Simulation KS models. In this experimental method the mixing processes are generated by the evolution of a discrete set of forced turbulent plumes. We describe the corresponding qualitative results and the quantitative conclusions based on measures of the density field and of the height of the fluid layers. We present an experimental analysis to characterize the partial mixing process. The conclusions of this analysis are related to the mixing efficiency and the height of the final mixed layer as functions of the Atwood number, which ranges from 9.8x10-3 to 1.34x 10-1.

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.