Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. © 2013 Aprile et al.FAA was recipient of a graduate fellowship from the Italian Ministry of Education, University and Research. AD is grateful for support from Murray Edwards College, Cambridge, through a Junior Research Fellowship. FS is a Sir Henry Wellcome Fellow. CR acknowledges financial support by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', through ISCIII with cofunding by FEDER (CP10/00527). JLPB is a Royal Society University Research Fellow. FAA and PT are grateful for support from Regione Lombardia (NEDD and >Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi>). CMD acknowledges support from BBSRC (BB/E019927/1), the Wellcome Trust (094425/Z/10/Z), the European Commission (project LSHM-CT-2006-037525). NC acknowledges support from Human Frontiers Science Program (HFSP) through a Long-term Fellowship (LT000795/2009).Peer Reviewe

Warping and F-term uplifting

We analyse the effective supergravity model of a warped compactification with matter on D3 and D7-branes. We find that the main effect of the warp factor is to modify the F-terms while leaving the D-terms invariant. Hence warped models with moduli stabilisation and a small positive cosmological constant resulting from a large warping can only be achieved with an almost vanishing D-term and a F-term uplifting. By studying string-motivated examples with gaugino condensation on magnetised D7-branes, we find that even with a vanishing D-term, it is difficult to achieve a Minkowski minimum for reasonable parameter choices. When coupled to an ISS sector the AdS vacua is uplifted, resulting in a small gravitino mass for a warp factor of order 10^-5.Comment: 24 pages, v3: typos, minor clarifications adde

Local Cooperativity in an Amyloidogenic State of Human Lysozyme Observed at Atomic Resolution

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein

Levofloxacin versus placebo for the prevention of tuberculosis disease in child contacts of multidrug-resistant tuberculosis: study protocol for a phase III cluster randomised controlled trial (TB-CHAMP)

Background Multidrug-resistant (MDR) tuberculosis (TB) presents a challenge for global TB control. Treating individuals with MDR-TB infection to prevent progression to disease could be an effective public health strategy. Young children are at high risk of developing TB disease following infection and are commonly infected by an adult in their household. Identifying young children with household exposure to MDR-TB and providing them with MDR-TB preventive therapy could reduce the risk of disease progression. To date, no trials of MDR-TB preventive therapy have been completed and World Health Organization guidelines suggest close observation with no active treatment. Methods The tuberculosis child multidrug-resistant preventive therapy (TB-CHAMP) trial is a phase III cluster randomised placebo-controlled trial to assess the efficacy of levofloxacin in young child contacts of MDR-TB cases. The trial is taking place at three sites in South Africa where adults with MDR-TB are identified. If a child aged < 5 years lives in their household, we assess the adult index case, screen all household members for TB disease and evaluate any child aged < 5 years for trial eligibility. Eligible children are randomised by household to receive daily levofloxacin (15–20 mg/kg) or matching placebo for six months. Children are closely monitored for disease development, drug tolerability and adverse events. The primary endpoint is incident TB disease or TB death by one year after recruitment. We will enrol 1556 children from approximately 778 households with an average of two eligible children per household. Recruitment will run for 18–24 months with all children followed for 18 months after treatment. Qualitative and health economic evaluations are embedded in the trial. Discussion If the TB-CHAMP trial demonstrates that levofloxacin is effective in preventing TB disease in young children who have been exposed to MDR-TB and that it is safe, well tolerated, acceptable and cost-effective, we would expect that that this intervention would rapidly transfer into policy. Trial registration ISRCTN Registry, ISRCTN92634082. Registered on 31 March 2016

Optimizing Hydroxychloroquine Dosing for Patients With COVID-19: An Integrative Modeling Approach for Effective Drug Repurposing.

Hydroxychloroquine (HCQ) is a promising candidate for Coronavirus disease of 2019 (COVID-19) treatment. The optimal dosing of HCQ is unknown. Our goal was to integrate historic and emerging pharmacological and toxicity data to understand safe and efficacious HCQ dosing strategies for COVID-19 treatment. The data sources included were (i) longitudinal clinical, pharmacokinetic (PK), and virologic data from patients with severe acute respiratory syndrome-2 (SARS-CoV-2) infection who received HCQ with or without azithromycin (n&nbsp;=&nbsp;116), (ii) in vitro viral replication data and SARS-CoV-2 viral load inhibition by HCQ, (iii) a population PK model of HCQ, and (iv) a model relating chloroquine PKs to corrected QT (QTc) prolongation. A mechanistic PK/virologic/QTc model for HCQ was developed and externally validated to predict SARS-CoV-2 rate of viral decline and QTc prolongation. SARS-CoV-2 viral decline was associated with HCQ PKs (P&nbsp;&lt;&nbsp;0.001). The extrapolated patient half-maximal effective concentration (EC50 ) was 4.7&nbsp;µM, comparable to the reported in vitro EC50s . HCQ doses &gt;&nbsp;400&nbsp;mg b.i.d. for ≥5&nbsp;days were predicted to rapidly decrease viral loads, reduce the proportion of patients with detectable SARS-CoV-2 infection, and shorten treatment courses, compared with lower dose (≤&nbsp;400&nbsp;mg daily) regimens. However, HCQ doses &gt;&nbsp;600&nbsp;mg b.i.d. were also predicted to prolong QTc intervals. This prolongation may have clinical implications warranting further safety assessment. Due to COVID-19's variable natural history, lower dose HCQ regimens may be indistinguishable from controls. Evaluation of higher HCQ doses is needed to ensure adequate safety and efficacy

Optimizing Hydroxychloroquine Dosing for Patients With COVID‐19: An Integrative Modeling Approach for Effective Drug Repurposing

Hydroxychloroquine (HCQ) is a promising candidate for Coronavirus disease of 2019 (COVID‐19) treatment. The optimal dosing of HCQ is unknown. Our goal was to integrate historic and emerging pharmacological and toxicity data to understand safe and efficacious HCQ dosing strategies for COVID‐19 treatment. The data sources included were (i) longitudinal clinical, pharmacokinetic (PK), and virologic data from patients with severe acute respiratory syndrome‐2 (SARS‐CoV‐2) infection who received HCQ with or without azithromycin (n = 116), (ii) in vitro viral replication data and SARS‐CoV‐2 viral load inhibition by HCQ, (iii) a population PK model of HCQ, and (iv) a model relating chloroquine PKs to corrected QT (QTc) prolongation. A mechanistic PK/virologic/QTc model for HCQ was developed and externally validated to predict SARS‐CoV‐2 rate of viral decline and QTc prolongation. SARS‐CoV‐2 viral decline was associated with HCQ PKs (P < 0.001). The extrapolated patient half‐maximal effective concentration (EC50) was 4.7 µM, comparable to the reported in vitro EC50s. HCQ doses 400 mg b.i.d. for ≥5 days were predicted to rapidly decrease viral loads, reduce the proportion of patients with detectable SARS‐CoV‐2 infection, and shorten treatment courses, compared with lower dose (≤ 400 mg daily) regimens. However, HCQ doses 600 mg b.i.d. were also predicted to prolong QTc intervals. This prolongation may have clinical implications warranting further safety assessment. Due to COVID‐19's variable natural history, lower dose HCQ regimens may be indistinguishable from controls. Evaluation of higher HCQ doses is needed to ensure adequate safety and efficacy.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEpu

Synthesis, crystal structure and magnetism of new salicylamidoxime-based hexanuclear manganese(III) single-molecule magnets.

International audienceSalicylamidoxime was used to synthesize 13 new polynuclear MnIII complexes. We present the crystallographic structures, the magnetic susceptibility and the magnetization measurements of eight of them (1–8) with the general formula [Mn6O2(H2N-sao)6(L)2(solvent)4–6] (L = carboxylate, chloride, 2-cyanophenolate; solvent = H2O, MeOH, EtOH, py). These complexes consist of two trinuclear {MnIII3(μ3-O)(H2N-sao)3}+ cationic units linked together via two oximate and two phenolate oxygen atoms. All behave as single-molecule magnets, with the spin ground state varying from 4 to 12 and anisotropy energy barriers from 24 to 86 K, the latter being as high as the present record barrier in the Mn6 complexes. DFT calculations were performed to compute the exchange magnetic coupling constants J between the metallic ions and to provide an orbital interpretation of exchange. Our results are in line with previously reported results with the parent salicylaldoxime derivatives. The Mn–N–O–Mn torsion angle appears as the main parameter controlling the J values. The critical angle where the exchange coupling between two MnIII switches from antiferromagnetic to ferromagnetic is 27°, less than the one found in related complexes with salicylaldoxime (30°). We propose a structural classification of the {Mn6} complexes in four classes depending on the coordination of the axial carboxylate. The work points out the structural flexibility of such systems, their sensitivity to solvent effects and their ability to achieve high anisotropy energy barriers by simple desolvation

Isolated 5′ Signals Are an Atypical Pattern To Be Considered as Positive for ALK Rearrangement: A Brief Report of Three Cases and Review of the Literature

Anaplastic lymphoma kinase (ALK) rearrangement is reported in 3% to 8% of patients with lung adenocarcinoma and can be detected by fluorescent in situ hybridization (FISH) or indirectly by immunohistochemistry. In FISH assay, isolated 5′ signal (loss of 3′ signal) is usually considered negative. We report three young nonsmoking patients with stage IV lung adenocarcinoma. Strong ALK expression in tumor cells detected by immunohistochemistry was observed in all cases, but FISH revealed an isolated 5′ signal pattern. Massive parallel “next-generation” sequencing was performed in two patients and confirmed ALK rearrangement. The three patients were treated and responded to crizotinib after 14, 10, and 31 months

Inhibition of α-Synuclein Fibril Elongation by Hsp70 Is Governed by a Kinetic Binding Competition between α-Synuclein Species.

The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.BBSRC (BB/J002119/1), European Research Council (337969

The different protein variants have different propensities to oligomerize.

<p>Analytical SEC and nESI mass spectra of the different protein constructs measured at the same protein concentration (6 µM): FL-Hsp70 (A, C), SBD641 (B, D), SBD556 (E, G) and C-term (F, H) variants. The SEC elution profiles were fitted to multi-peak Gaussian functions to evaluate the relative fractions of monomeric and oligomeric protein species. The molecular masses of the standard proteins used to calibrate the SEC column are reported at the top of the chromatogram. The elution peaks in SEC and the peaks in MS (Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067961#pone.0067961.s001" target="_blank">File S1</a>) corresponding to monomeric protein are coloured in green for all the variants, while those for dimers are in red, trimers in blue and tetramers in yellow. Note that comparing visually the SEC and MS data quantitatively is difficult as the relative peak heights in the mass spectra report on molecular abundance, which is independent of oligomeric state, while the SEC data depend on molecular absorbance.</p