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The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells. Further, phospho-ATP5F1A (p-Y-ATP5F1A) prevented its binding to its physiological inhibitor, ATP5IF1 (ATP synthase inhibitory factor subunit 1), causing sustained mitochondrial activity to promote cancer cell growth. TNK2 inhibitor, (R)-9b reversed this process and induced mitophagy-based autophagy to mitigate prostate tumor growth while sparing normal prostate cells. Further, depletion of p-Y-ATP5F1A was needed for (R)-9b-mediated mitophagic response and tumor growth. Moreover, Tnk2 transgenic mice displayed increased p-Y-ATP5F1A and loss of mitophagy and exhibited formation of prostatic intraepithelial neoplasia (PINs). Consistent with these data, a marked increase in p-Y-ATP5F1A was seen as prostate cancer progressed to the malignant stage. Overall, this study uncovered the molecular intricacy of tyrosine kinase-mediated mitochondrial energy regulation as a distinct cancer cell mitochondrial vulnerability and provided evidence that TNK2 inhibitors can act as "mitocans" to induce cancer-specific mitophagy. Abbreviations: ATP5F1A: ATP synthase F1 subunit alpha; ATP5IF1: ATP synthase inhibitory factor subunit 1; CRPC: castration-resistant prostate cancer; DNM1L: dynamin 1 like; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; Mut-ATP5F1A: Y243,246A mutant of ATP5F1A; OXPHOS: oxidative phosphorylation; PC: prostate cancer; PINK1: PTEN induced kinase 1; p-Y-ATP5F1A: phosphorylated tyrosine 243 and 246 on ATP5F1A; TNK2/ACK1: tyrosine kinase non receptor 2; Ub: ubiquitin; WT: wild type [ABSTRACT FROM AUTHOR]

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

In the past two decades, 7 coronaviruses have infected the human population, with two major outbreaks caused by SARS-CoV and MERS-CoV in the year 2002 and 2012, respectively. Currently, the entire world is facing a pandemic of another coronavirus, SARS-CoV-2, with a high fatality rate. The spike glycoprotein of SARS-CoV-2 mediates entry of virus into the host cell and is one of the most important antigenic determinants, making it a potential candidate for a vaccine. In this study, we have computationally designed a multi-epitope vaccine using spike glycoprotein of SARS-CoV-2. The overall quality of the candidate vaccine was validated in silico and Molecular Dynamics Simulation confirmed the stability of the designed vaccine. Docking studies revealed stable interactions of the vaccine with Toll-Like Receptors and MHC Receptors. The in silico cloning and codon optimization supported the proficient expression of the designed vaccine in E. coli expression system. The efficiency of the candidate vaccine to trigger an effective immune response was assessed by an in silico immune simulation. The computational analyses suggest that the designed multi-epitope vaccine is structurally stable which can induce specific immune responses and thus, can be a potential vaccine candidate against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design. [ABSTRACT FROM AUTHOR]

The proton pore of the F1Fo ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state. Structures are here presented of the c10 ring from Saccharomyces cerevisiae determined at pH 8.3, 6.1 and 5.5, at resolutions of 2.0 Å, 2.5 Å and 2.0 Å, respectively. The overall structure of this mitochondrial c-ring is similar to known homologs, except that the essential carboxylate, Glu59, adopts an open extended conformation. Molecular dynamics simulations reveal that opening of the essential carboxylate is a consequence of the amphiphilic nature of the crystallization buffer. We propose that this new structure represents the functionally open form of the c subunit, which facilitates proton loading and release. [ABSTRACT FROM AUTHOR]

The crystal structure of yeast mitochondrial F1 ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the α3β3 sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F1 ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F1 ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F1 ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis. [ABSTRACT FROM AUTHOR]

A genetically modified (His6-tagged) form of the mitochondrial F1 -ATPase (MW = 370 kDa) has been purified from the yeast Saccharomyces cerevisiae and crystallized in the presence of polythelene glycol (PEG) 6000 as a precipitant, 1 mM NiCl2, 1mM Mg AMP-PNP and 50 μM Mg ADP. X-ray diffraction data were obtained on three separate occasions using synchrotron radiation, with a progression in the quality of the diffraction data, which improved from 3.3 to 3.0 to 2.8 &Aring . On the second occasion, the diffraction was improved by a crystal-annealing procedure. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 110.6, b = 294.2, c = 190.4 Å, β = 101.6°. The asymmetric unit contains three molecules of yeast F1, with a corresponding volume per protein weight (VM) of 2.8 ų Da-1 and a solvent content of 55%. [ABSTRACT FROM AUTHOR]

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]

Examines the complementation of deletion mutants in the genes encoding the F(sub 1)-ATPase. Expression of the corresponding bovine subunits in yeast Saccharomyces cerevisiae; Construction of the null mutants; Expression constructs generation.

The F1-ATPase is a multimeric enzyme (α3β3γδε) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding α-, β-, γ- or δ-subunits were unable to grow, while the strain with a null mutation in ε was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in γ or δ became 100% ρ0/ρ­ and the strain with the null mutation in γ grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: ΔαΔγ, ΔβΔγ, Δatp11Δγ, ΔαΔδ, ΔβΔγ or Δatp11Δδ. These results indicate that ε is not an essential component of the ATP synthase and that mutations in the genes encoding the α- and β-subunits and in ATP11 are epistatic to null mutations in the genes encoding the γ- and δ-subunits. These data suggest that the propensity to form ρ0/ρ­ mutations in the γ and δ null deletion mutant stains and the slow growing phenotypes of the null γ mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells. [ABSTRACT FROM AUTHOR]

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197→Ser mutation in the β subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high, The Thr197→Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197→Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197→Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197→Ser mutation as measured by determining the KiADP values for multi-site ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under mulfisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wildtype and mutant enzymes and the Thr197→Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197→Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197→Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197→Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197→Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP. [ABSTRACT FROM AUTHOR]

Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo. Luo, Zhou et al. show that Bedaquiline (BDQ, Sirturo), approved to treat multi-drug-resistant tuberculosis, inhibits the yeast and human mitochondrial ATP synthases in addition to its intended target, the Mycobacterium tuberculosis ATP synthase. The structure of the mitochondrial ATP synthase bound to BDQ suggests a means to modify this inhibitor to increase its specificity for the M. tuberculosis enzyme, thereby reducing its side effects for patients. [ABSTRACT FROM AUTHOR]

The unicellular green alga Chlamydomonas reinhardtii is a biflagellated cell with two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Here, we probe how actin redundancy contributes to flagellar assembly. Disrupting a single actin allows complete flagellar assembly. However, when disrupting both actins using latrunculin B (LatB) treatment on the nap1 mutant background, we find that actins are necessary for flagellar growth from newly synthesized limiting flagellar proteins. Under total actin disruption, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles. We also find that there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins. • At least one filamentous actin required for full flagellar assembly • Transition zone flagellar gate affected upon actin disruption • Actin required for normal flagellar protein synthesis • Golgi-adjacent vesicles accumulate upon total actin loss Prevailing knowledge tells us that intracellular trafficking of flagellar proteins occurs on microtubule tracks. In this study, Jack et al. discover that filamentous actin is required for full flagellar assembly, normal flagellar protein synthesis, an intact flagellar gating region, and vesicle transport in Chlamydomonas reinhardtii. [ABSTRACT FROM AUTHOR]