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Supplement Figure 2. Mitochondria isolated from ZR-75-1 cells and assayed for PRODH activity by NADH induction to compare three propargyl- analogs with N-PPG for potential PRODH inhibitory activity.

Supplement Figure 1. Mitochondria isolated from ZR-75-1 cells and assayed for PRODH activity assessed by either P5C production (A) or substrate inducing NADH formation (B), with use of NADH bioassay to compare PRODH inhibitor effects (C, D).

To demonstrate that the oral N-PPG treatment (treated flies in Suppl. Fig 3B panel A) phenocopies the Sluggish-A phenotype of SlgA null mutant flies as previously described (18), geotaxis of untreated SlgA mutant flies is video illustrated (right tube) and compared to that of untreated wildtype flies (left tube).

Legend for Supplementary videos S1A and S1B

Supplement Figure 3. Confocal images of control and N-PPG treated ZR-75-1 cell cultures show absence of mitochondrial membrane recruitment of PINK/PARKIN complex (A), despite positive induction of mitochondrial GRP-75 import (B), within 24 h of N-PPG treatment.

Advanced hepatic fibrosis, driven by the activation of hepatic stellate cells (HSCs), affects millions worldwide and is the strongest predictor of mortality in nonalcoholic steatohepatitis (NASH); however, there are no approved antifibrotic therapies. To identify antifibrotic drug targets, we integrated progressive transcriptomic and morphological responses that accompany HSC activation in advanced disease using single-nucleus RNA sequencing and tissue clearing in a robust murine NASH model. In advanced fibrosis, we found that an autocrine HSC signaling circuit emerged that was composed of 68 receptor-ligand interactions conserved between murine and human NASH. These predicted interactions were supported by the parallel appearance of markedly increased direct stellate cell-cell contacts in murine NASH. As proof of principle, pharmacological inhibition of one such autocrine interaction, neurotrophic receptor tyrosine kinase 3–neurotrophin 3, inhibited human HSC activation in culture and reversed advanced murine NASH fibrosis. In summary, we uncovered a repertoire of antifibrotic drug targets underlying advanced fibrosis in vivo. The findings suggest a therapeutic paradigm in which stage-specific therapies could yield enhanced antifibrotic efficacy in patients with advanced hepatic fibrosis. This is all of the human data samples and analysis for the paperAn autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis. GEO data here: GSE212837 The folderliver_data_cellranger_all.zipcontains the raw and filtered cellranger output for every sample. This allows reprocessing of all cells including the use of methods that use empty droplets to calculate ambient RNA. The folderAutocrine_liver_paper_jupyter_notebooks.zipcontains all of the notebooks used for analysis: Autocrine_signaling_liver_data_merge_all_select_celltype_subsets.ipynb is the initial noteboook for processing all datasets and merging. It also contains in the first cell instructions for creating a conda environment that will allow all of the tools to run. There are 5 notebooks for qc and cleaning of each celltype: Autocrine_signaling_liver_data_Stellate_cell_recluster.ipynb,Autocrine_signaling_liver_data_Hepatocyte_cell_recluster.ipynb, Autocrine_signaling_liver_data_Endo_cell_recluster.ipynb,Autocrine_signaling_liver_data_Cholangiocyte_cell_recluster.ipynb, and Autocrine_signaling_liver_data_NKTcell_cell_recluster.ipynb There is a final notebook for re-merging the cleaned celltype objects and final clustering, DE and analysis:Autocrine_signaling_liver_data_recluster_all_cleaned_celltypes_analysis.ipynb The fileraw_all_cellranger.h5ad.gzis all cells passing cellranger filter annotated with celltype and ambient RNA and doublet scoring, but otherwise no cells or genes have been removed. The foldercelltype_subsets_v1.zipcontains the roughly filtered celltypes for each of the celltypes. The foldercleaned_celltype_subsets_for_merge.zipcontains the final qc'd and filtered celltypes that are merged for final analysis. It also contains the final merged and batch normalized object. The python scriptcellphone_db_liver_all_cells_clean.pycontains a script for running cellphoneDB on final data.

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery (ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community., Aging, 14 (2), ISSN:1945-4589

An increasing aging population poses a significant challenge to societies worldwide. A better understanding of the molecular, cellular, organ, tissue, physiological, psychological, and even sociological changes that occur with aging is needed in order to treat age-associated diseases. The field of aging research is rapidly expanding with multiple advances transpiring in many previously disconnected areas. Several major pharmaceutical, biotechnology, and consumer companies made aging research a priority and are building internal expertise, integrating aging research into traditional business models and exploring new go-to-market strategies. Many of these efforts are spearheaded by the latest advances in artificial intelligence, namely deep learning, including generative and reinforcement learning. To facilitate these trends, the Center for Healthy Aging at the University of Copenhagen and Insilico Medicine are building a community of Key Opinion Leaders (KOLs) in these areas and launched the annual conference series titled "Aging Research and Drug Discovery (ARDD)" held in the capital of the pharmaceutical industry, Basel, Switzerland (www.agingpharma.org). This ARDD collection contains summaries from the 6th annual meeting that explored aging mechanisms and new interventions in age-associated diseases. The 7th annual ARDD exhibition will transpire 2nd-4th of September, 2020, in Basel.

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.

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA., Aging, 12 (24), ISSN:1945-4589

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.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single‐gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age‐related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long‐lived animals with elevated Jun‐N‐terminal Kinase signaling. We find that these animals exhibit a delayed age‐related decline in protein turnover rates, as well as decreased steady‐state neuronal glucose‐6‐phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose‐6‐phosphate dehydrogenase (G6PD). Over‐expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long‐lived mutants.

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

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.

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., The molecular mechanisms of mitochondrial dysfunction in the premature ageing Werner syndrome were elusive. Here the authors show that NAD+ depletion-induced impaired mitophagy contributes to this phenomenon, shedding light on potential therapeutics.

Significance Mutations of mtDNA accumulate in aging humans and other mammals to cause mitochondrial dysfunction in a subset of cells in various tissues. Furthermore, experimental induction of mtDNA mutations causes a premature aging syndrome in the mouse. To study if mitochondrial dysfunction is universally involved in shortening life span in metazoans, we generated a series of fruit fly lines with varying levels of mtDNA mutations. Unexpectedly, we report that fruit flies are remarkably tolerant to mtDNA mutations, as exemplified by their lack of effect on physiology and lifespan. Only an artificially induced, very drastic increase of the mtDNA mutation load will lead to reduced lifespan, showing that mtDNA mutations are unlikely to limit lifespan in natural fruit fly populations., Mammals develop age-associated clonal expansion of somatic mtDNA mutations resulting in severe respiratory chain deficiency in a subset of cells in a variety of tissues. Both mathematical modeling based on descriptive data from humans and experimental data from mtDNA mutator mice suggest that the somatic mutations are formed early in life and then undergo mitotic segregation during adult life to reach very high levels in certain cells. To address whether mtDNA mutations have a universal effect on aging metazoans, we investigated their role in physiology and aging of fruit flies. To this end, we utilized genetically engineered flies expressing mutant versions of the catalytic subunit of mitochondrial DNA polymerase (DmPOLγA) as a means to introduce mtDNA mutations. We report here that lifespan and health in fruit flies are remarkably tolerant to mtDNA mutations. Our results show that the short lifespan and wide genetic bottleneck of fruit flies are limiting the extent of clonal expansion of mtDNA mutations both in individuals and between generations. However, an increase of mtDNA mutations to very high levels caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan under standard conditions. In addition, the effects of dietary restriction, widely considered beneficial for organismal health, were attenuated in flies with very high levels of mtDNA mutations.

The mitochondrial unfolded protein response (UPRmt) has been associated with long lifespan across metazoans. In Caenorhabditis elegans, mild developmental mitochondrial stress activates UPRmt reporters and extends lifespan. We show that similar developmental stress is necessary and sufficient to extend Drosophila lifespan, and identify Phosphoglycerate Mutase 5 (PGAM5) as a mediator of this response. Developmental mitochondrial stress leads to activation of FoxO, via Apoptosis Signal-regulating Kinase 1 (ASK1) and Jun-N-terminal Kinase (JNK). This activation persists into adulthood and induces a select set of chaperones, many of which have been implicated in lifespan extension in flies. Persistent FoxO activation can be reversed by a high-protein diet in adulthood, through mTORC1 and GCN-2 activity. Accordingly, the observed lifespan extension is prevented on a high-protein diet and in FoxO-null flies. The diet-sensitivity of this pathway has important implications for interventions that seek to engage the UPRmt to improve metabolic health and longevity.

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.

Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, “S1QELs”). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 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-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

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.

Mitochondria play a central role in the aging process. Studies in model organisms have started to integrate mitochondrial effects on aging with the maintenance of protein homeostasis. These findings center on the mitochondrial unfolded protein response (UPR mt ), which has been implicated in lifespan extension in worms, flies, and mice, suggesting a conserved role in the long-term maintenance of cellular homeostasis. Here, we review current knowledge of the UPR mt and discuss its integration with cellular pathways known to regulate lifespan. We highlight how insight into the UPR mt is revolutionizing our understanding of mitochondrial lifespan extension and of the aging process.

In vivo studies have reported preferential partitioning of Ras GTPases into ordered lipid-protein membrane domains, a process believed to regulate both cellular signaling and protein trafficking.1 However studies in vitro have failed to quantify a preferential partitioning of full length Ras proteins into the liquid ordered phase2,3 and thus a biophysically validated mechanism for in vivo sorting of Ras is still missing. We recently showed that lipidated proteins localize to highly curved membranes in vitro.4 Here we study both in vitro and in vivo whether recruitment by membrane curvature can sort full length lipid-anchored Ras.We employ a single vesicle fluorescence based assay to quantify in vitro the sorting by membrane curvature of full-length Ras proteins. We demonstrate a more than 50 fold increase in protein density on membranes of high curvature as compared to the density on flat membranes. To test for recruitment by membrane curvature in vivo we utilize hypo-osmotic swelling of cells, which flattens curved membrane regions. By measuring the local protein density using FRET,5 we detect a significant reduction in the clustering of Ras and other lipidated proteins upon membrane flattening. This demonstrates that recruitment by membrane curvature can sort Ras and potentially other lipidated proteins in cellular membranes. Furthermore sorting by membrane curvature constitutes the first biophysical sorting mechanism for Ras validated by both in vitro and in vivo measurements.1 Hancock, J. F. Nat. Rev. Mol. Cell Biol.4 (2003).2 Johnson, S. A. et al.Biochim. Biophys. Acta - Biomembranes1798 (2010).3 Nicolini, C. et al.J. Am. Chem. Soc.128 (2006).4 Hatzakis, N. S. et al.Nat. Chem. Biol.5 (2009).5 Kohnke, M. et al.Chem. Biol.19 (2012).

In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output and hence is important to recombinant and, most certainly, endogenous gene expression.

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.

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.

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.