Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Thermophilic enzymes can operate at higher temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule F\"orster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.Comment: 14 pages, 7 figure

A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif

Abstract: The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. Author Summary: Essential cellular processes such as biosynthesis, transport, and motility are sustained by the energy released in the hydrolysis of ATP, the universal energy carrier in living cells. Most ATP in the cell is produced by a membrane-bound enzyme, the ATP synthase, through a rotary mechanism that is coupled to the translocation of ions across the membrane. The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons (proton-motive force), but a number of organisms, including some important human pathogens, use gradients of sodium ions instead (sodium-motive force). The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex, the c-ring, which is the smallest known biological rotor. The functional mechanism of the rotor ring and its variations among different organisms are of wide interest, because of this enzyme's impact on metabolism and disease, and because of its potential for nanotechnology applications. Here, we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is implicated in periodontal diseases. We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach, combining cell-growth and biochemical assays, X-ray crystallography and computer-simulation methods. Two crystal structures of the membrane rotor were solved, at low and high pH, revealing an atypical ion-recognition motif mediated by two carboxylate side-chains. This motif is shared by other human pathogens, such as Mycobacterium tuberculosis or Streptococcus pneumonia, whose ATP synthases are targets of novel antibiotic drugs. The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F. nucleatum were thus examined. Our results provide the basis for future pharmacological efforts against this important pathogen

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

A simple retroelement based knock-down system in Dictyostelium

Gefördert durch den Publikationsfonds der Universität Kasse

Schematic presentation of the knock-down system.

<p>(A) A gene fragment is inserted in between the left and right ITR as a trigger sequence. (B) Genes used for knock-down analysis are presented as mRNA with accession number and sequence length. Trigger sequences are indicated by black bars with position of the first and last nucleotide in respect to the ATG start. Oligonucleotides and their orientation used as hybridization probes are indicated by black arrows.</p

Analysis of ITR promoter activity.

<p>(A) The GFP coding region was inserted between actin15 promoter (A15P) and actin8 terminator (A8T), DIRS–1 left ITR (lITR) and A8T, inverted DIRS-1 right ITR (rITR) and A8T. (B) Detection of GFP mRNA expression in Ax2 wild type driven by the constructs depicted in Fig 1A. As a loading control, the blot was rehybridised with a ³²P labelled oligonucleotide directed against actin (left panel). Western blot shows GFP protein expression in Ax2 wild type driven by the constructs depicted in Fig 1A. Coronin A (CorA) was used as a loading control (middle panel). The right panel shows the corresponding quantification of Western blots. Analysis was performed using ImageJ. Ax2 A15 GFP: n = 8. Ax2 lITR GFP: n = 4. Ax2 rITR GFP: n = 4. Error bars: mean with SD. (C) Detection of mRNA expression (left) and protein level (middle) for GFP driven by the lITR in Ax2 wild type and agnA knock-out (agnA-) cell lines. Numbers in square brackets indicate numbers of independent populations. The right panel shows the corresponding quantification of Western blots. Analysis was performed using ImageJ. Similar results were obtained with the right ITR (data not shown). Ax2 lITR GFP: n = 4. agnA- lITR GFP: n = 4. Error bars: mean with SD.</p

Comparison of published knock-out phenotypes with knock-downs by ITR constructs.

<p>(A) Nuclei count in Ax2 wild type cells and different knock-down strains. Knock-down of corA (lTR corA rITR) resulted in increased multinuclearity. 200 cells of each strain of two independent transformations were counted. (B) Knock-down of mhcA by an ITR construct showed huge cells containing up to 30 nuclei and more compared to Ax2 wild type and to a control knock-down of abpA. Scale bar 50μm. (C) Growth curve of Ax2 wild type cells and different knock-down strains. Knock-down of casK resulted in a slow growing culture. Measurement of cell density in the indicated cell lines over 72 hours. Data are plotted as relative cell count over time. Ax2, lITR GFP rITR, lITR abpA rITR: n = 2; lITR casK rITR: n = 4.</p

Determination of 5’ endgroups by Terminator 5'-phosphate-dependent exonuclease (5'exo).

<p>RNA samples of the ITR GFP strains (silencing <i>in cis</i>) and ITR abpA strains (silencing <i>in trans</i>) were digested with 5'exo, in buffer A and buffer B as indicated, separated on an acrylamide gel, blotted and probed with radiolabelled oligonucleotides complementary to 5.8S (digestion control), tRNA^Asp (control for undigested RNA) and for GFP and abpA siRNAs respectively.</p