A mechano-chemical model for energy transduction in cytochrome c oxidase: the work of a Maxwell's god

Cytochrome c3 has a central role in the energetics of Desulfovibrio sp., where it performs an electroprotonic energy transduction step. This process uses a network of cooperativities, largely based on anti-Coulomb components, resulting from a mechano-chemical energy coupling mechanism. This mechanism provides a model coherent with the data available for the redox chemistry of haem a of cytochrome c oxidase and its link to the activation of protons. A crucial feature of the model is an anti-Coulomb effect that sets the stage for a molecular ratchet, ensuring vectoriality for the redox-driven localised movement of protons across the membrane, against an electrochemical gradient.     

Protein Clusters in Phosphotyrosine Signal Transduction

Signal transduction systems based on tyrosine phosphorylation are central to cell–cell communication in multicellular organisms. Typically, in such a system, the signal is initiated by activating tyrosine kinases associated with transmembrane receptors, which induces tyrosine phosphorylation of the receptor and/or associated proteins. The phosphorylated tyrosines then serve as docking sites for the binding of various downstream effector proteins. It has long been observed that the cooperative association of the receptors and effectors produces higher-order protein assemblies (clusters) following signal activation in virtually all phosphotyrosine signal transduction systems. However, mechanistic studies on how such clustering processes affect signal transduction outcomes have only emerged recently. Here we review current progress in decoding the biophysical consequences of clustering on the behavior of the system, and how clustering affects how these receptors process information.     

Understanding the cooperative interaction between myosin II and actin cross-linkers mediated by actin filaments during mechanosensation

Myosin II is a central mechanoenzyme in a wide range of cellular morphogenic processes. Its cellular localization is dependent not only on signal transduction pathways, but also on mechanical stress. We suggest that this stress-dependent distribution is the result of both the force-dependent binding to actin filaments and cooperative interactions between bound myosin heads. By assuming that the binding of myosin heads induces and/or stabilizes local conformational changes in the actin filaments that enhances myosin II binding locally, we successfully simulate the cooperative binding of myosin to actin observed experimentally. In addition, we can interpret the cooperative interactions between myosin and actin cross-linking proteins observed in cellular mechanosensation, provided that a similar mechanism operates among different proteins. Finally, we present a model that couples cooperative interactions to the assembly dynamics of myosin bipolar thick filaments and that accounts for the transient behaviors of the myosin II accumulation during mechanosensation. This mechanism is likely to be general for a range of myosin II-dependent cellular mechanosensory processes.     

How cooperative are protein folding and unfolding transitions?

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding.     

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.     

Molecular mechanisms of cytolysis induced by human lymphokine-activated killer cells

It has been shown that lysis of tumor target cells caused by lymphokine-activated killers is possible both upon a direct contact and in the presence of isolated nongranular cytotoxic proteins. The contact of cytolytic lymphocytes with K-562 cells leads to Fas L activation on the lymphocyte membrane and secretion of a broad spectrum of soluble cytotoxic proteins immunologically related to Tag 7 described earlier. These proteins can form inactive complexes, which are reactivated upon heating and addition of ATP. The proteins induced discrete cytolytic processes in tumor cells, differing in the rate of cytolysis and the mechanism of the apoptotic signal transduction. Fast processes (revealed in 3 h) mediated by caspases, and slow ones (in 24 h) with the supposed involvement of mitochondria were detected. A scheme for the lymphokine-activated killer interaction with target tumor cells is proposed.     

Activation of p21ras by transforming growth factor beta in epithelial cells.

The transforming growth factor beta (TGF beta) family members are ubiquitously expressed and control a variety of cellular processes by interacting with at least two types of high affinity cell surface receptors. However, the primary signal transduction mechanism of the receptors is unknown. The ras-encoded 21-kDa GTP binding proteins have recently been shown to mediate the effects of other polypeptide growth factors. Here we show that both TGF beta 1 and TGF beta 2 (5 ng/ml) result in a rapid (within 6 or 12 min, respectively) stimulation of GTP bound to p21ras in TGF beta-sensitive intestinal epithelial cells. Further, the CCL64 epithelial cell line, extremely sensitive to growth inhibition by TGF beta, displayed a concentration-dependent increase in GTP bound to p21ras by TGF beta 1 and a rapid activation of p21ras by TGF beta 2. The results provide the first direct evidence for rapid activation of a receptor coupling component for TGF beta in epithelial cells.     

Structural bioenergetics and energy transduction mechanisms.

Life depends on transduction processes that couple cellular metabolism to environmental energy sources such as light or reduced compounds. These primary energy sources must be efficiently converted into forms that can be utilized by cells for biosynthesis, motility, transport, regulation, and other metabolic functions. In recent years, there has been an explosive increase in the determination of structures for proteins mediating energy transduction processes. These developments provide the opportunity to evaluate the structural basis for the efficient coupling of two energetic processes, which defines the area of structural bioenergetics. Here, we present some general features of energy transduction processes, including arguments that effective coupling of two processes by a transduction protein occurs by way of conformational states that are common to the catalysis of each process. This is illustrated by examples from the nucleotide switch family of proteins, with emphasis on the nitrogenase system where ATP hydrolysis is coupled to an electron transfer reaction.     

Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network

Dynamic rearrangements of the actin cytoskeleton, following T-cell antigen receptor (TCR) engagement, provide the structural matrix and flexibility to enable intracellular signal transduction, cellular and subcellular remodeling, and driving effector functions. Recently developed cutting-edge imaging technologies have facilitated the study of TCR signaling and its role in actin-dependent processes. In this review, we describe how TCR signaling cascades induce the activation of actin regulatory proteins and the formation of actin networks, and how actin dynamics is important for T-cell homeostasis, activation, migration, and other effector functions.     

P(II) signal transducers: novel functional and structural insights

The P(II) signal transduction proteins have key functions in coordinating the regulation of central metabolic processes. Signals from the carbon, nitrogen and energy status of the cells are converted into different conformational (and modification) states of the P(II) proteins. Depending on these states, the P(II) proteins interact with various target proteins, most of which perform or regulate crucial reactions in nitrogen assimilatory pathways. This review presents recent progress in the elucidation of novel P(II) functions and in gaining novel structural insights into how the signals convert the P(II) states and how the activity of targets is affected by P(II) interaction.     

Kinetic timing: a novel mechanism that improves the accuracy of GTPase timers in endosome fusion and other biological processes

The GTPase superfamily contains a large number of proteins that function as molecular switches by binding and hydrolyzing GTP molecules. They are localized at various intracellular organelles and control diverse cellular processes. For many GTPases, the lifetime of the activated, GTP-bound state is believed to serve as a timer in determining the activation time of a biological event such as membrane fusion and signal transduction. However, such a timer is intrinsically stochastic due to thermal noise at the level of single GTPase molecules. Here, we describe a mathematical model that shows how a directional GTPase cycle, in a nonequilibrium steady-state driven by GTP hydrolysis, can significantly reduce the variance in the lifetime of an activated GTPase molecule and thereby increase the accuracy and efficiency of the timer. This mechanism, termed kinetic timing, articulates a clear function for the energy consumption in GTPase-controlled biological processes. It provides a rationale for why biological timers utilize a GTP hydrolysis cycle rather than a simple GTP binding–dissociation equilibrium, and why the GTP-bound state is a better timer than the GDP-bound state. It also explains the necessity for the existence of multiple GTP-bound intermediates identified by fluorescence spectroscopy and nuclear magnetic resonance studies.     

The EF-hand domain: A globally cooperative structural unit

EF-hand Ca(2+)-binding proteins participate in both modulation of Ca(2+) signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca(2+). The EF-hand domains of calbindin D(9k) and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D(9k) to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.     

The angiotensin Ⅱ type 1 receptor and receptor-associated proteins

INTRODUCTIONThe renin-angiotensin system (RAS) is consid-ered to be the major regulator of blood pressure)electrolyte balance and renal, neuronal as well as en-docrine functions related to cardiovascular control.The RAS is the key factor in most cases essential hy-pertension, as indicated by successes in treatment ofhypertensive patients with various angiotensin I con-verting enzyme (ACE) inhibitors and receptor block-ers. Renin was a central subject of intense investigation because of…     

The angiotensin Ⅱ type 1 receptor and receptor－associated proteins

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.     

o-GlcNAc transferase is activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization in NG-108-15 cells

Post-translational modification of cellular proteins by beta-o-linked N-acetylglucosamine (o-GlcNAc) moieties plays a significant role in signal transduction by modulating protein stability, protein-protein interactions, transactivation processes, and the enzyme activities of target proteins. Though various classes of proteins are known to be regulated by o-GlcNAc modification (o-GlcNAcylation), the mechanism that regulates o-linked GlcNAc transferase (OGT) activity remains unknown. Here, we report that potassium chloride-induced depolarization provokes the activation of OGT and subsequent o-GlcNAcylation of proteins in neuroblastoma NG-108-15 cells. Moreover, such an induction of protein o-GlcNAcylation was abolished by treating cells with either a voltage-gated calcium channel inhibitor or a calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. In addition, CaMKIV was found to specifically phosphorylate and activate OGT in vivo and in vitro, which implies that CaMKIV is required for depolarization-induced activation of OGT. Furthermore, we found that OGT is involved in depolarization-induced and CaMKIV-dependent activation of activator protein-1 (AP-1) and subsequent tissue inhibitor of metalloproteinase-1 (Timp-1) gene expression. Taken together, our findings suggest that CaMKIV activated OGT, and OGT has an essential role on the process of CaMKIV-dependent AP-1 activation under depolarization in neuronal cells.