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Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo. Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG–like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

Summary Using high-throughput screening we identified small molecules that suppress superoxide and/or H 2 O 2 production during reverse electron transport through mitochondrial respiratory complex I (site I Q ) without affecting oxidative phosphorylation (suppressors of site I Q electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site I Q is a normal contributor to mitochondrial superoxide-H 2 O 2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H 2 O 2 production by site I Q is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H 2 O 2 production by site I Q is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site I Q to cell physiology and pathology and have great potential as therapeutic leads.

We have investigated the mobility of two EGFP-tagged DNA repair proteins, WRN and BLM. In particular, we focused on the dynamics in two locations, the nucleoli and the nucleoplasm. We found that both WRN and BLM use a "DNA-scanning" mechanism, with rapid binding-unbinding to DNA resulting in effective diffusion. In the nucleoplasm WRN and BLM have effective diffusion coefficients of 1.62 and 1.34 μm(2)/s, respectively. Likewise, the dynamics in the nucleoli are also best described by effective diffusion, but with diffusion coefficients a factor of ten lower than in the nucleoplasm. From this large reduction in diffusion coefficient we were able to classify WRN and BLM as DNA damage scanners. In addition to WRN and BLM we also classified other DNA damage proteins and found they all fall into one of two categories. Either they are scanners, similar to WRN and BLM, with very low diffusion coefficients, suggesting a scanning mechanism, or they are almost freely diffusing, suggesting that they interact with DNA only after initiation of a DNA damage response.

RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations. These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase-dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease.

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.

Key points The present study utilized a novel method aiming to investigate mitochondrial function in human skeletal muscle at submaximal levels and at a predefined membrane potential. The effect of age and training status was investigated using a cross-sectional design. Ageing was found to be related to decreased leak regardless of training status. Increased training status was associated with increased mitochondrial hydrogen peroxide emission. Abstract Despite numerous studies, there is no consensus about whether mitochondrial function is altered with increased age. The novelty of the present study is the determination of mitochondrial function at submaximal activity rates, which is more physiologically relevant than the ex vivo functionality protocols used previously. Muscle biopsies were taken from 64 old or young male subjects (aged 60–70 or 20–30 years). Aged subjects were recruited as trained or untrained. Muscle biopsies were used for the isolation of mitochondria and subsequent measurements of DNA repair, anti-oxidant capacity and mitochondrial protein levels (complexes I–V). Mitochondrial function was determined by simultaneous measurement of oxygen consumption, membrane potential and hydrogen peroxide emission using pyruvate + malate (PM) or succinate + rotenone (SR) as substrates. Proton leak was lower in aged subjects when determined at the same membrane potential and was unaffected by training status. State 3 respiration was lower in aged untrained subjects. This effect, however, was alleviated in aged trained subjects. H2O2 emission with PM was higher in aged subjects, and was exacerbated by training, although it was not changed when using SR. However, with a higher manganese superoxide dismuthase content, the trained aged subjects may actually have lower or similar mitochondrial superoxide emission compared to the untrained subjects. We conclude that ageing and the physical activity level in aged subjects are both related to changes in the intrinsic functionality of the mitochondrion in skeletal muscle. Both of these changes could be important factors in determining the metabolic health of the aged skeletal muscle cell.

A series of 1-(10,11-dihydrodibenzo[b,f]thiepin-10-yl)-4-methylpiperazine analogues substituted in the 8-position of the 10,11-dihydrodibenzo[b,f]thiepine scaffold with aryl, heteroaryl, amine, and amide substituents are described. The compounds were designed using the previously reported Liljefors-Bøgesø pharmacophore model for dopamine D(2) and α(1)-adrenoceptor antagonists, with the aim of obtaining selective α(1)-adrenoceptor antagonists suitable for development as radioligands for imaging of central α(1)-adrenoceptors by positron emission tomography. Sixteen aryl and heteroaryl substituted octoclothepin analogues were prepared by a convergent synthesis via coupling of 1-methyl-4-(8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-10,11-dihydrodibenzo[b,f]thiepin-10-yl)piperazine with aryl and heteroaryl halides under palladium catalysis. The most selective compound obtained, (S)-N-((11-(4-methylpiperazin-1-yl)-10,11-dihydrodibenzo[b,f]thiepin-2-yl)methyl)isobutyramide (S)-35, showed a similar subnanomolar affinity compared to α(1a), α(1b), and α(1d)-adrenoceptors and a selectivity ratio of 20, 440, and 20 with respect to D(2), 5-HT(2C), and H(1) receptors, respectively.

Sorting and trafficking of membrane-anchored Ras GTPases is critical for signaling and is believed to rely on their preferential portioning in ordered lipid-protein membrane domains (1). However studies in vitro have failed to quantify the preferential partitioning of full length Ras proteins into the liquid ordered phase(2), indicating that a physical principle underlying sorting of Ras is missing. We recently showed that lipidated proteins localize to highly curved membranes in vitro(3, 4). Here we provide a mechanistic insight on how membrane curvature can drive N-Ras sorting.Combining the results of our in vitro assays, measurements on single vesicles, with in vivo studies, hypo-osmotic swelling of cells that flattens curved membrane regions, revealed that : a) N-Ras is preferentially recruited in areas of high membrane curvature and b) membrane curvature is the enabling factor underlying the selective partitioning of NRas in ordered domains. The combined readout of mean field theory calculations and coarse grain simulations provided a mechanistic insight on preferential partitioning in highly curved areas, via the changes in lateral pressure of the outer monolayer when curving an ordered versus a disordered membrane. In addition to providing the first biophysical sorting mechanism for Ras validated by both in vitro and in vivo measurements, our data indicate that membrane curvature may act as a generic cue underlying trafficking and sorting of multiple lipidated proteins.References1. J. F. Hancock, Nat. Rev. Mol. Cell Biol. 4, 373 (2003).2. S. A. Johnson Biochim. Biophys. Acta - Biomembranes 1798, 1427 (2010).3. N. S. Hatzakis et al., Nat. Chem. Biol. 5, 835 (2009).4. V. K. Bhatia, et al Semin. Cell Dev. Biol. 21, 381 (2010).

Cellular membranes define cell boundaries and provide active means for proteins transport and compartmentalization to certain organelles. The prevailing mechanism underlying lipidated protein transport and sorting is based on their selective upconcentration in transient membrane domains of altered fluidity, termed “raft domains”. However the majority of biophysical studies have failed to report increased partition to the lo phase1. We recently illustrated that the farnesylated Gβγ subunit of G protein upconcentrates in highly curved areas and introduced the notion that membrane remodeling may act as a cue for regulating lipidated protein cellular distribution2.Here we capitalized on our developed nanorrays of surface tethered liposomes to quantify the precise effect of membrane curvature and domain formation on the redistribution of the abundant class of signaling proteins, the Ras2-4. Our results demonstrate for the first time that NRas proteins upconcentrate in highly curved areas showing ∼10 fold increased densities as compared to flat bilayers. We furthermore found that NRas does not selectively bind in flat raft domains as compared to ld phases. When however the lo “raft domains” where combined with high curvature a remarkable upconcentration ∼80 fold was documented in the highly curved areas. These findings indicate that lo domains enhance the intrinsic ability of membranes to promote lateral organization of lipidated proteins in highly curved areas.References1. Silvious JR., Biochim. Biophys. Acta, 1610, 174 (2003).2. Hatzakis, N.S. et al. How Curved Membranes Recognize Amphipathic Helices and Protein Anchoring Motifs. Nat. Chem. Biol. 5, 835 (2009).3. Christensen, S., Bolinger P-Y., Hatzakis NS., Stamou D., Nat Nanotech (2011) In Press.4. Larsen K J, Hatzakis N S. Stamou D., J Amer Chem Soc 2011, 133, 10685.