Structural basis of the allosteric trigger of the Hsp70 chaperone proteins.

This work solves a decades-old dilemma that stood in the way of understanding the allosteric mechanism of Hsp70 (heat shock 70 kDa) chaperone proteins. Hsp70s are central to protein folding, refolding, and trafficking in organisms ranging from Archae to Homo Sapiens, both at normal and at stressed cellular conditions. Hsp70s are comprised of two main domains: a 44 kDa N-terminal nucleotide-binding domain (NBD), and a 25 kDa substrate-binding domain (SBD) that harbors the substrate binding site. The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. It has long been a goal of structural biology to characterize the nature of the allosteric coupling in these proteins. However, even the most sophisticated X-ray crystallography studies of the isolated NBD could show no difference in overall conformation between the ATP and ADP state. Hence the dilemma: how is the state of the nucleotide communicated between NBD and SBD? The solution of the dilemma is especially interesting in light of the fact that Hsp70s are ancient proteins, and amongst the first allosteric proteins in nature.Here we report a solution NMR study of the NBD of the Hsp70 from Thermus thermophilus, in the APO, ADP and AMP-PNP states, where the latter is a non-hydrolysable ATP analogue. Using the modern NMR methods of residual dipolar coupling analysis, we discovered that the nucleotide binding cleft opens up by as much as 20 degrees between the AMP-PNP (closed) and ADP (open) state. We also discover that a surface cleft, hypothesized to be essential for the allosteric coupling between NBD and SBD, echoes these changes. Hence, the nature of the allosteric trigger and coupling for Hsp70 chaperones is revealed here for the first time, solving the dilemma

Behavior and Impact of Zirconium in the Soil–Plant System: Plant Uptake and Phytotoxicity

Because of the large number of sites they pollute, toxic metals that contaminate terrestrial ecosystems are increasingly of environmental and sanitary concern (Uzu et al. 2010, 2011; Shahid et al. 2011a, b, 2012a). Among such metals is zirconium (Zr), which has the atomic number 40 and is a transition metal that resembles titanium in physical and chemical properties (Zaccone et al. 2008). Zr is widely used in many chemical industry processes and in nuclear reactors (Sandoval et al. 2011; Kamal et al. 2011), owing to its useful properties like hardness, corrosion-resistance and permeable to neutrons (Mushtaq 2012). Hence, the recent increased use of Zr by industry, and the occurrence of the Chernobyl and Fukashima catastrophe have enhanced environmental levels in soil and waters (Yirchenko and Agapkina 1993; Mosulishvili et al. 1994 ; Kruglov et al. 1996)

Combination therapy with oral treprostinil for pulmonary arterial hypertension. A double-blind placebo-controlled clinical trial

Rationale: Oral treprostinil improves exercise capacity in patients with pulmonary arterial hypertension (PAH), but the effect on clinical outcomes was unknown. Objectives: To evaluate the effect of oral treprostinil compared with placebo on time to first adjudicated clinical worsening event in participants with PAH who recently began approved oral monotherapy. Methods: In this event-driven, double-blind study, we randomly allocated 690 participants (1:1 ratio) with PAH to receive placebo or oral treprostinil extended-release tablets three times daily. Eligible participants were using approved oral monotherapy for over 30 days before randomization and had a 6-minute-walk distance 150 m or greater. The primary endpoint was the time to first adjudicated clinical worsening event: death; hospitalization due to worsening PAH; initiation of inhaled or parenteral prostacyclin therapy; disease progression; or unsatisfactory long-term clinical response. Measurements and Main Results: Clinical worsening occurred in 26% of the oral treprostinil group compared with 36% of placebo participants (hazard ratio, 0.74; 95% confidence interval, 0.56–0.97; P = 0.028). Key measures of disease status, including functional class, Borg dyspnea score, and N-terminal pro–brain natriuretic peptide, all favored oral treprostinil treatment at Week 24 and beyond. A noninvasive risk stratification analysis demonstrated that oral treprostinil–assigned participants had a substantially higher mortality risk at baseline but achieved a lower risk profile from Study Weeks 12–60. The most common adverse events in the oral treprostinil group were headache, diarrhea, flushing, nausea, and vomiting. Conclusions: In participants with PAH, addition of oral treprostinil to approved oral monotherapy reduced the risk of clinical worsening. Clinical trial registered with www.clinicaltrials.gov (NCT01560624)

Association of Immunosuppression and Human Immunodeficiency Virus (HIV) Viremia with Anal Cancer Risk in Persons Living with HIV in the United States and Canada

Background: People living with human immunodeficiency virus (HIV; PLWH) have a markedly elevated anal cancer risk, largely due to loss of immunoregulatory control of oncogenic human papillomavirus infection. To better understand anal cancer development and prevention, we determined whether recent, past, cumulative, or nadir/peak CD4+ T-cell count (CD4) and/or HIV-1 RNA level (HIV RNA) best predict anal cancer risk. Methods: We studied 102 777 PLWH during 1996-2014 from 21 cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. Using demographics-adjusted, cohort-stratified Cox models, we assessed associations between anal cancer risk and various time-updated CD4 and HIV RNA measures, including cumulative and nadir/peak measures during prespecified moving time windows. We compared models using the Akaike information criterion. Results: Cumulative and nadir/peak CD4 or HIV RNA measures from approximately 8.5 to 4.5 years in the past were generally better predictors for anal cancer risk than their corresponding more recent measures. However, the best model included CD4 nadir (ie, the lowest CD4) from approximately 8.5 years to 6 months in the past (hazard ratio [HR] for <50 vs ≥500 cells/μL, 13.4; 95% confidence interval [CI], 3.5-51.0) and proportion of time CD4 <200 cells/μL from approximately 8.5 to 4.5 years in the past (a cumulative measure; HR for 100% vs 0%, 3.1; 95% CI, 1.5-6.6). Conclusions: Our results are consistent with anal cancer promotion by severe, prolonged HIV-induced immunosuppression. Nadir and cumulative CD4 may represent useful markers for identifying PLWH at higher anal cancer risk

Improving experimentation and interpretation in solution nuclear magnetic resonance investigations.

In the past three decades, roughly 100 fundamentally different Nuclear Magnetic Resonance (NMR) experiments have been developed for the determination of protein and nucleic acid structures, interactions, dynamics and kinetics. These experiments are now widely and successfully used in structural biology, proteomics, bioinformatics, metabolomics, and pharmaceutical sciences. However, in seeking to quantitatively understand biomolecules one must consider that the NMR experimentation may perturb the molecular biophysical quantity sought. Moreover, limitations on available instrument time unfavorably affect the validity of statistical analysis of the physical quantities obtained. Some of these limitations and concerns are considered and explored herein. In NMR relaxation experimentation, sample heating caused by the applied NMR pulses themselves is a major problem that can seriously bias the values of the dynamical parameters derived. A compensation and saturation scheme for constant sample heating is proposed and statistically evaluated. It is concluded that the scheme allows for reproducible, robust and rapid acquisition of NMR spin relaxation data sets. Molecular dynamics parameters derived from transverse Carr-Purcell-Meiboom-Gill relaxation experiments are seriously affected by the NMR frequency setting with respect to the protein's resonance frequencies. A novel phase cycle scheme is proposed and statistically evaluated; it is shown that it can greatly improve the relative reliability of these measured rates. Experimental Residual Dipolar Coupling (RDC) and structural data for large proteins have a high degree of uncertainty because of the intrinsic low sensitivity of the experiments. Herein it is systematically explored how these errors propagate through commonly used RDC analysis software and how they affect the derived structural and dynamical quantities. The RDC analysis is demonstrated to be remarkably robust even in unfavorable cases of much structural and experimental uncertainty. It is concluded that the RDC method is valid and powerful towards elucidating orientational information on large biomolecular systems and complexes. The issues described are just a few imperfections in NMR spectroscopy that are addressed in this thesis. These investigations help to address the confidence and reliability of conclusions drawn from these NMR techniques.Ph.D.Biological SciencesBiophysicsPhysical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126337/2/3238126.pd