Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

BACKGROUND: The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA) is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET) uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2), a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. RESULTS: TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2). The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. CONCLUSION: The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function

Ras oncogene-independent activation of RALB signaling is a targetable mechanism of escape from NRAS(V12) oncogene addiction in acute myeloid leukemia.

Somatic mutations that lead to constitutive activation of NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML

R-spondin 2 drives liver tumor development in a yes-associated protein-dependent manner

202012 bcrcVersion of RecordPublishe

A new material for prevention of peritendinous fibrotic adhesions after tendon repair: oxidised regenerated cellulose (Interceed), an absorbable adhesion barrier

In this experimental study, we aimed to examine the ability of absorbable oxidised regenerated cellulose (Interceed, TC-7, Johnson & Johnson, USA) to inhibit the formation of peritendinous fibrotic adhesions after tendon repair in rats. Both Achilles tendons of 23 female Wistar-Albino rats weighing between 350 and 450 grams were cut and repaired. On the right side, Interceed absorbable adhesion barriers were wrapped around the repaired tendon (group I). On the left, the same procedures were applied except for the Interceed wrapping and these were grouped as control (group II). Animals were sacrificed at postoperative day 28 and macroscopic and histological examination was performed. All the animals survived and no tendon rupture was observed. No wound dehiscence, wound infection or exposure of repaired tendons occurred. Macroscopically, there were three (13.1%) tendons without adhesion formation and 20 (86.9%) tendons with inferior adhesion formation in group I; on the other hand, there were 16 tendons (69.5%) with medium grade adhesion formation and seven tendons (30.5%) with severe peritendinous adhesion formation in group II (control group) (p < 0.05). Histologically, adhesion formation was absent in 11 tendons (47.8%) and slight in 12 tendons (52.2%) in group I; while in group II, it was slight in two (8.6%), moderate in 15 (65.2%) and severe in six tendons (26.2%) (p < 0.05). Sixteen (69.5%) of 23 tendons in group I and 11 (47.8%) of 23 tendons in group II showed no inflammatory reaction (p  < 0.05). Nineteen (82.6%) tendons in group I and only one tendon in group II showed excellent to good tendon healing (p = 0.00). According to our results, we feel that Interceed may have an intraoperative role to play in the reduction of adhesions after surgical tendon repair. This study suggests that absorbable oxidised regenerated cellulose merits further evaluation as a potential treatment to inhibit the formation of peritendinous adhesions. Rigorous and extensive controlled trials should be undertaken on patients undergoing tendon repair with or without this barrier

Allosteric activation transitions in enzymes and biomolecular motors: insights from atomistic and coarse-grained simulations

The chemical step in enzymes is usually preceded by a kinetically distinct activation step that involves large-scale conformational transitions. In simple enzymes this step corresponds to the closure of the active site; in more complex enzymes, such as biomolecular motors, the activation step is more complex and may involve interactions with other biomolecules. These activation transitions are essential to the function of enzymes and perturbations in the scale and/or rate of these transitions are implicated in various serious human diseases; incorporating key flexibilities into engineered enzymes is also considered a major remaining challenge in rational enzyme design. Therefore it is important to understand the underlying mechanism of these transitions. This is a significant challenge to both experimental and computational studies because of the allosteric and multi-scale nature of such transitions. Using our recent studies of two enzyme systems, myosin and adenylate kinase (AK), we discuss how atomistic and coarse-grained simulations can be used to provide insights into the mechanism of activation transitions in realistic systems. Collectively, the results suggest that although many allosteric transitions can be viewed as domain displacements mediated by flexible hinges, there are additional complexities and various deviations. For example, although our studies do not find any evidence for cracking in AK, our results do underline the contribution of intra-domain properties (e.g., dihedral flexibility) to the rate of the transition. The study of mechanochemical coupling in myosin highlights that local changes important to chemistry require stabilization from more extensive structural changes; in this sense, more global structural transitions are needed to activate the chemistry in the active site. These discussions further emphasize the importance of better understanding factors that control the degree of co-operativity for allosteric transitions, again hinting at the intimate connection between protein stability and functional flexibility. Finally, a number of topics of considerable future interest are briefly discussed