1515 Background: Older adults receiving chemotherapy (CT) are at a high risk of toxicity (Tox) and of functional decline. Promptly acting upon CT Tox in older adults is difficult, particularly in ...developing countries where triage systems and personnel are limited. We previously showed that monitoring older patients using an accelerometer-equipped smartphone is feasible, and that a decline in the number of steps/day may represent a marker of Tox. We aimed to evaluate the diagnostic accuracy of an objective patient-centered measure of physical function (steps/day) for the remote detection of Tox in older adults receiving CT. Methods: We included consecutive patients aged ≥65 years starting first line CT for solid tumors at a single center in Mexico City. Patients underwent a geriatric assessment and were provided with an accelerometer-equipped smartphone. Daily steps were recorded for ≥7 days pre-CT initiation, and median number of pre-CT steps/day was calculated. Patients with ≤600 median pre-CT steps/day, and those using walking aids, were excluded (16% of recruited patients). Steps were monitored daily, compared with median pre-CT steps/day, and % decline was calculated. Patients were called daily, and Tox was assessed using PRO-CTCAE questionnaires. The % decline in steps/day from pre-CT was considered as the index test for Tox, while patient report was considered the reference standard. The association between % step decline and moderate/severe Tox was examined using generalized linear-mixed models. AUC was calculated and Youden’s index used to choose cutoff points for Tox detection. Results: 116 patients were included (96 development cohort, 20 validation cohort). Median age was 73y (range 65-91), 55% were female, and 65% had ≤high school education. The most common tumors were colon (21.5%), pancreas (17.5%), and gastric (12%). 28% of participants had never used a smartphone. The median number of pre-CT steps/day was 2979. Patients were followed for 6764 days, with Tox detected on 64.4% of days. Moderate/severe self-reported toxicity was detected on 1245 days (22.1%), while mild/no toxicity was detected on 4378 days (77.9%). AUC analysis for the development cohort demonstrated that a 32% decrease in steps/day from pre-CT median showed a sensitivity of 77.6% and a specificity of 67.3% for detecting moderate/severe Tox. Sensitivity and specificity of the cutoff in the validation cohort were 75.8% and 69.6%, respectively. Tox associated with a decline in steps/day included fatigue, pain, and nausea. Conclusions: A decline in the number of steps/day measured using an accelerometer-equipped smartphone was useful for the remote detection of moderate/severe Tox in older adults with cancer receiving CT, with a high sensitivity and specificity. This patient-centric measure could be used in clinical practice and research to detect and act promptly on Tox and, potentially, improve outcomes.
Abstract only
Ligand‐induced disorder‐to‐order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered domains. Here, we present data on an RTX ...(« Repeat in ToXin ») protein, RC
L
, an intrinsically disordered protein (IDP) that folds upon calcium binding. RTX motifs are calcium‐binding nonapeptide sequences that are found in more than 250 virulence factors secreted by Gram‐negative pathogenic bacteria. The RC
L
protein is a RTX region of the CyaA toxin, one of the major virulence factors secreted by Bordetella pertussis, the causative agent of the whooping cough. Using a combination of biophysical approaches, we showed that RC
L
exhibits the hallmarks of IDP in the absence of calcium. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of RC
L
. We further showed that the crowding agent Ficoll70 did not affect the structural content of the apo‐state and holo‐state of RC
L
but increased protein affinity for calcium and stabilizes both states of RC
L
, increasing their half‐melting temperature (ΔTm), without affecting enthalpy changes. The power law dependence of the ΔTm increase on the volume fraction allowed the estimation of the Flory exponents of the thermally unfolded states. Altogether, our data suggest that, in the apo‐state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the secretion machinery. Subsequently, both calcium gradient across bacterial cell wall and molecular crowding enhance the calcium‐dependent folding and stability of RTX proteins once secreted in the extracellular milieu.
Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure ...of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg338, Asn347, and Asp360) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn347; ACm1A), two (Arg338 and Asp360; ACm2A), or three residues (Arg338, Asn347, and Asp360; ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3A·C-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.
Background: Adenylyl cyclase (AC) from Bordetella pertussis is activated when it interacts with calmodulin (CaM).
Results: A triple mutant of AC, which was predicted by molecular modeling, exhibited a highly reduced affinity for CaM.
Conclusion: This study suggests that a long range connection between CaM and the AC catalytic loop is crucial for AC activation.
Significance: Molecular modeling identified critical molecular determinants for the allosteric activation of AC.
The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA ...invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.
•CyaA is a major virulent factor produced by Bordetella pertussis, the causative agent of whooping cough.•Disorder-to-order transitions are central to CyaA toxin biogenesis.•CyaA is adapted to the diversity of calcium concentrations encountered during the successive steps of the intoxication process.
Under physiological conditions, intrinsically disordered proteins (IDPs) are unfolded, mainly because of their low hydrophobicity and the strong electrostatic repulsion between charged residues of ...the same sign within the protein. Softwares have been designed to facilitate the computation of the mean net charge of proteins (formally protein valence) from their amino acid sequences. Nevertheless, discrepancies between experimental and computed valence values for several proteins have been reported in the literature. Hence, experimental approaches are required to obtain accurate estimation of protein valence in solution. Moreover, ligand-induced disorder-to-order transition is involved in the folding of numerous IDPs. Some of the ligands are cations or anions, which, upon protein binding, decrease the mean net charge of the protein, favoring its folding via a charge reduction effect. An accurate determination of the mean net charge of protein in both its ligand-free intrinsically disordered state and in its folded, ligand-bound state allows one to estimate the number of ligands bound to the protein in the holo-state. Here, we describe an experimental protocol to determine the mean net charge of protein, from its electrophoretic mobility, its molecular mass and its hydrodynamic radius.
Size exclusion chromatography coupled online to a Tetra Detector Array in combination with analytical ultracentrifugation (or with quasi-elastic light scattering) is a useful methodology to ...characterize hydrodynamic properties of macromolecules, including intrinsically disordered proteins. The time-averaged apparent hydration and the shape factor of proteins can be estimated from the measured parameters (molecular mass, intrinsic viscosity, hydrodynamic radius) by these techniques. Here we describe in detail this methodology and its application to characterize hydrodynamic and conformational changes in proteins.
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