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Survival according to BRAF-V600 tumor mutations : an analysis of 437 patients with primary melanoma
(2014)
The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.
F1Fo‐ATP synthase is one of the best studied macromolecular machines in nature. It can be inhibited by a range of small molecules, which include the polyphenols, resveratrol and piceatannol. Here, we introduce Photoswitchable Inhibitors of ATP Synthase, termed PIAS, which were synthetically derived from these polyphenols. They can be used to reversibly control the enzymatic activity of purified yeast Yarrowia lipolyticaATP synthase by light. Our experiments indicate that the PIAS bind to the same site in the ATP synthase F1 complex as the polyphenols in their trans form, but they do not bind in their cis form. The PIAS could be useful tools for the optical precision control of ATP synthase in a variety of biochemical and biotechnological applications.
We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle. Formal Correction: This article has been formally corrected to address the following errors. 1. The images for Figures S2 and S3 were incorrectly switched. The image that appears as Figure S2 should be Figure S3, and the image that appears as Figure S3 should be Figure S2. The figure legends appear in the correct order. Please view the correct... (read formal correction) 2. The images for Figures S2 and S3 were incorrectly switched. The image that appears as Figure S2 should be Figure S3, and the image that appears as Figure S3 should be Figure S2. The figure legends appear in the correct order. Please view the correct... (read formal correction)
A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif
(2013)
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.
Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.
Background: Uveal melanoma (UM) is highly refractory to treatment with dismal prognosis in advanced stages. The value of the combined checkpoint blockade with CTLA-4 and PD-1 inhibition in metastatic UM is currently unclear.
Methods: Patients with metastatic or unresectable UM treated with ipilimumab in combination with a PD-1 inhibitor were collected from 16 German skin cancer centers. Patient records of 64 cases were analyzed for response, progression-free survival (PFS), overall survival (OS), and safety. Clinical parameters and serum biomarkers associated with OS and treatment response were determined with Cox regression modelling and logistic regression.
Results: The best overall response rate to combined checkpoint blockade was 15.6% with 3.1 and 12.5% complete and partial response, respectively. The median duration of response was 25.5 months (range 9.0–65.0). Stable disease was achieved in 21.9%, resulting in a disease control rate of 37.5% with a median duration of the clinical benefit of 28.0 months (range 7.0–65.0). The median PFS was 3.0 months (95% CI 2.4–3.6). The median OS was estimated to 16.1 months (95% CI 12.9–19.3). Regarding safety, 39.1% of treated patients experienced a severe, treatment-related adverse event according to the CTCAE criteria (grade 3: 37.5%; grade 4: 1.6%). The most common toxicities were colitis (20.3%), hepatitis (20.3%), thyreoiditis (15.6%), and hypophysitis (7.8%). A poor ECOG performance status was an independent risk factor for decreased OS (p = 0.007).
Conclusions: The tolerability of the combined checkpoint blockade in UM may possibly be better than in trials on cutaneous melanoma. This study implies that combined checkpoint blockade represents the hitherto most effective treatment option available for metastatic UM available outside of clinical trials.
Highlights
• Cryo-EM structure of a yeast F1Fo-ATP synthase dimer
• Inhibitor-free X-ray structure of the F1 head and rotor complex
• Mechanism of ATP generation by rotary catalysis
• Structural basis of cristae formation in the inner mitochondrial membrane
Summary
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.