Organelle-specific Proteins Research Articles

MamF-like proteins are distant Tic20 homologs involved in organelle assembly in bacteria

Organelle-specific protein translocation systems are essential for organelle biogenesis and maintenance in eukaryotes but thought to be absent from prokaryotic organelles. Here, we demonstrate that MamF-like proteins are crucial for the formation and functionality of bacterial magnetosome organelles. Deletion of mamF-like genes in the Alphaproteobacterium Magnetospirillum gryphiswaldense results in severe defects in organelle positioning, biomineralization, and magnetic navigation. These phenotypic defects result from the disrupted targeting of a subset of magnetosomal proteins that contain C-terminal glycine-rich integral membrane domains. Phylogenetic analyses reveal an ancient evolutionary link between MamF-like proteins and plastidial Tic20. Our findings redefine the molecular roles of MamF-like proteins and suggest that organelle-specific protein targeting systems also play a role in bacterial organelle formation.

Peroxisomal import stress activates integrated stress response and inhibits ribosome biogenesis.

Impaired organelle-specific protein import triggers a variety of cellular stress responses, including adaptive pathways to balance protein homeostasis. Most of the previous studies focus on the cellular stress response triggered by misfolded proteins or defective protein import in the endoplasmic reticulum or mitochondria. However, little is known about the cellular stress response to impaired protein import in the peroxisome, an understudied organelle that has recently emerged as a key signaling hub for cellular and metabolic homeostasis. To uncover evolutionarily conserved cellular responses upon defective peroxisomal import, we carried out a comparative transcriptomic analysis on fruit flies with tissue-specific peroxin knockdown and human HEK293 cells expressing dominant-negative PEX5C11A. Our RNA-seq results reveal that defective peroxisomal import upregulates integrated stress response (ISR) and downregulates ribosome biogenesis in both flies and human cells. Functional analyses confirm that impaired peroxisomal import induces eIF2α phosphorylation and ATF4 expression. Loss of ATF4 exaggerates cellular damage upon peroxisomal import defects, suggesting that ATF4 activation serves as a cellular cytoprotective mechanism upon peroxisomal import stress. Intriguingly, we show that peroxisomal import stress decreases the expression of rRNA processing genes and inhibits early pre-rRNA processing, which leads to the accumulation of 47S precursor rRNA and reduction of downstream rRNA intermediates. Taken together, we identify ISR activation and ribosome biogenesis inhibition as conserved adaptive stress responses to defective peroxisomal import and uncover a novel link between peroxisomal dysfunction and rRNA processing.

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1.

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15th and 21st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31st to 40th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41st to 50th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.

Circulating primitive murine erythroblasts undergo complex proteomic and metabolomic changes during terminal maturation

AbstractPrimitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for the growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semisynchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome, and lipidome of primitive erythroblasts isolated from embryonic day (E) 10.5 and E12.5 of mouse gestation, representing their transition from basophilic erythroblast to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterizations of these precursors have highlighted a transition toward the expression of protein elements characteristic of mature red blood cell structure and function. Our analysis confirmed a loss of organelle-specific protein components involved in messenger RNA processing, proteostasis, and metabolism. In parallel, we observed metabolic rewiring toward the pentose phosphate pathway, glycolysis, and the Rapoport-Luebering shunt. Activation of the pentose phosphate pathway in particular may have stemmed from increased expression of hemoglobin chains and band 3, which together control oxygen-dependent metabolic modulation. Increased expression of several antioxidant enzymes also indicated modification to redox homeostasis. In addition, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles.

Circulating Primitive Erythroblasts in the Murine Embryo Undergo Complex Proteomic and Metabolomic Changes during Terminal Maturation

Primitive erythropoiesis is a critical component of the fetal cardiovascular network and is essential for growth and survival of the mammalian embryo. The need to rapidly establish a functional cardiovascular system in the post-implantation murine embryo is met, in part, by the intravascular circulation of primitive erythroid precursors that mature as a single semi-synchronous cohort. To better understand the processes that regulate erythroid precursor maturation, we analyzed the proteome, metabolome and lipidome of primitive erythroblasts isolated from embryonic day (E)10.5 and E12.5 of mouse gestation, which represents their transition from basophilic erythroblast (BasoE) to orthochromatic erythroblast (OrthoE) stages of maturation. Previous transcriptional and biomechanical characterization of these precursors has highlighted a transition towards the expression of protein elements characteristic of mature red blood cell structure and function, including initial formation of a functional erythroid-specific cytoskeleton. Our analysis confirmed a loss of organelle-specific protein components - from mRNA maturation to translation, from metabolism to protein degradation - including ribosome, spliceosome, mitochondria and proteasomes. In parallel, we observed a metabolic rewiring towards the pentose phosphate pathway, glycolysis and the Rapoport-Luebering shunt, which is consistent with the progressively decreasing steady state levels of carboxylic acids, acylcarnitines and free fatty acids along with decreased mitochondrial content. In addition, activation of the pentose phosphate pathway in particular may stem from increases in the expression of mature red blood cell components including hemoglobin chains and band 3 that control oxygen-dependent metabolic modulation. Furthermore, this metabolic reprogamming may reflect increased control of oxidative stress in line with increased expression of several antioxidant enzymes, including catalase, peroxiredoxins and glutathione peroxidase 4. On the other hand, accumulation of oxylipins and cholesteryl esters in primitive OrthoE cells was paralleled by increased transcript levels of the p53-regulated cholesterol transporter (ABCA1) and decreased transcript levels of cholesterol synthetic enzymes including mevalonate kinase (MVK) and phosphomevalonate kinase (PVMK), highlighting transcriptional control of metabolic reprogramming during this developmental process. The present study characterizes the extensive metabolic rewiring that occurs in primary embryonic erythroid precursors as they prepare to enucleate and continue circulating without internal organelles. DisclosuresNemkov: Omix Thecnologies: Other: Co-founder. Hansen: Omix Technologies: Other: Co-founder; ScalmiBio: Membership on an entity's Board of Directors or advisory committees. Palis: Rubius Therapeutics: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Nanosims Tools and Probes for Imaging Intracellular Structures

In eukaryotic cells, intracellular lipid membranes act as barriers that segregate specific chemical reactions into organelles for increased biochemical efficiency. Organelle efficiency is maintained by the regulation of cholesterol and different lipid distributions within these organelles and is tightly controlled by intracellular transport and lipid metabolism. Disturbances in intracellular lipid trafficking, biosynthesis and degradation are hallmarks of fatal diseases involving an accumulation of cholesterol or lipids in endosomes and lysosomes, such as Niemann Pick Type II disease. Efforts to elucidate the mechanisms for lipid-mediated cellular (dys)function would benefit from an imaging technique that enables assessing the relative abundances of cholesterol and lipid species of interest between organelles. Towards this aim, we developed a secondary ion mass spectrometry (SIMS) approach to image the sphingolipid and cholesterol distributions within intact cells in three dimensions with a NanoSIMS instrument. Because the components of interest must produce secondary ions that contain distinctive elements or isotopes to permit their detection with a NanoSIMS instrument, organelle-specific labels must be incorporated into the cells to enable identifying specific organelles. This work describes a strategy that utilizes small protein tags and functionalized substrates that permit identify organelles of interest with NanoSIMS, and the development of data processing tools that produce images of these organelles at the proper depth in the 3D SIMS images. We created a transfected cell line that stably expresses enzymatic proteins fused to organelle-specific proteins. These proteins covalently attach to functionalized small-molecule substrates which can be detected with NanoSIMS and fluorescence microscopy. By combining these tools for organelle detection with lipid-specific metabolic isotope incorporation, semi-quantitative information concerning the abundance of cholesterol or specific lipids within organelles of interest may be obtained.

Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge

BackgroundWhole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail.ResultsIn this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression (R2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles.ConclusionsOur study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome.

Protein folding state-dependent sorting at the Golgi apparatus.

In eukaryotic cells, organelle-specific protein quality control (PQC) is critical for maintaining cellular homeostasis. Despite the Golgi apparatus being the major protein processing and sorting site within the secretory pathway, how it contributes to PQC has remained largely unknown. Using different chemical biology-based protein unfolding systems, we reveal the segregation of unfolded proteins from folded proteins in the Golgi. Quality control (QC) substrates are subsequently exported in distinct carriers, which likely contain unfolded proteins as well as highly oligomerized cargo that mimic protein aggregates. At an additional sorting step, oligomerized proteins are committed to lysosomal degradation, while unfolded proteins localize to the endoplasmic reticulum (ER) and associate with chaperones. These results highlight the existence of checkpoints at which QC substrates are selected for Golgi export and lysosomal degradation. Our data also suggest that the steady-state ER localization of misfolded proteins, observed for several disease-causing mutants, may have different origins.

Critical damage to the cellular organelles of Saccharomyces cerevisiae under sublethal conditions upon high pressure carbon dioxide treatment

ABSTRACTHigh pressure carbon dioxide treatment is a non-thermal pasteurization technique. However, critical damage, resulting from the treatment, to microbial cells has not been observed directly, and the detailed mechanism of the microbicidal activity is not understood. In this study, we analyzed the damage to Saccharomyces cerevisiae organelles, which were visualized using organelle-specific GFP fusion proteins. Yeast strains were subjected to high pressure carbon dioxide treatments at 30°C and 4.0 MPa for 2–8 h. Reduction in yeast cell viability was accompanied by the disruption of endoplasmic reticulum, nuclear membrane, Golgi body, and nucleolus. However, visible damage to the cell membrane was not observed. Fluorescence microscopy was utilized to confirm that high pressure carbon dioxide treatment damaged membranes of major organelles, but not the cell membrane.

Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells.

Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-specific proteins. Isolated enriched secretory granules were then used for affinity purification with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was confirmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunofluorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon “interactome.”

Rsp5 Ubiquitin ligase–mediated quality control system clears membrane proteins mistargeted to the vacuole membrane

Maintenance of organelle identity is profoundly dependent on the coordination between correct targeting of proteins and removal of mistargeted and damaged proteins. This task is mediated by organelle-specific protein quality control (QC) systems. In yeast, the endocytosis and QC of most plasma membrane (PM) proteins requires the Rsp5 ubiquitin ligase and ART adaptor network. We show that intracellular adaptors of Rsp5, Ear1, and Ssh4 mediate recognition and vacuolar degradation of PM proteins that escape or bypass PM QC systems. This second tier of surveillance helps to maintain cell integrity upon heat stress and protects from proteotoxicity. To understand the mechanism of the recognition of aberrant PM cargos by Ssh4-Rsp5, we mistarget multiple PM proteins de novo to the vacuolar membrane. We found that Ssh4-Rsp5 can target and ubiquitinate multiple lysines within a restricted distance from the membrane, providing a fail-safe mechanism for a diverse cargo repertoire. The mistargeting or misfolding of PM proteins likely exposes these lysines or shifts them into the "ubiquitination zone" accessible to the Ssh4-Rsp5 complex.

The Craft of Peroxisome Purification-A Technical Survey Through the Decades.

Purification technologies are one of the working horses in organelle proteomics studies as they guarantee the separation of organelle-specific proteins from the background contamination by other subcellular compartments. The development of methods for the separation of organelles was a major prerequisite for the initial detection and characterization of peroxisome as a discrete entity of the cell. Since then, isolated peroxisomes fractions have been used in numerous studies in order to characterize organelle-specific enzyme functions, to allocate the peroxisome-specific proteome or to unravel the organellar membrane composition. This review will give an overview of the fractionation methods used for the isolation of peroxisomes from animals, plants and fungi. In addition to "classic" centrifugation-based isolation methods, relying on the different densities of individual organelles, the review will also summarize work on alternative technologies like free-flow-electrophoresis or flow field fractionation which are based on distinct physicochemical parameters. A final chapter will further describe how different separation methods and quantitative mass spectrometry have been used in proteomics studies to assign the proteome of PO.

Abstract 1201: Disrupted endoplasmic reticulum-mitochondria contacts promote multidrug resistance

Abstract Background: Most high-risk neuroblastoma patients succumb to lethal therapy resistant disease acquired during the course of intensive multimodality treatment. This acquired therapy resistance is largely attributed to insensitivity to drug-induced apoptosis, however the exact mechanisms remain unknown. Apart from integrating death signals, mitochondria (mito) interact with the endoplasmic reticulum (ER) at close contact sites known as mitochondria associated membranes (MAM) of the ER to regulate calcium and lipid transfer and apoptotic sensitivity, a process often derailed in therapy resistant cancers. ER-mito contact sites are juxtaposed by various tethering protein complexes that include MFN2 and PACS2. Pathologic deregulation of these contact sites has been implicated in the genesis of neurodegenerative and metabolic disorders. Here, we show that disruption of bona fide ER-mito tethering proteins in therapy sensitive neuroblastomas induces apoptotic insensitivity and a drastic shift towards a resistance-like phenotype. Methods and Results: Previously we showed that isolated mitochondria from tumors at relapse resist induction of mitochondrial apoptosis when primed with the terminal death effectors that are downstream of therapeutic stress (tBid and Bim-BH3 peptide). We isolated mitochondria from seven matched isogenic tumor pairs obtained at diagnosis (DX) and relapse (REL) and quantified their apoptotic response to tBid and Bim by measuring cytochrome C release by ELISA. Electron microscopy (EM) image analyses of ER-mito contact sites revealed that REL tumors contain up to 70% fewer ER-mito interactions than their matched DX tumors, as confirmed by IB for organelle-specific proteins. Here, we recapitulate the post-therapy resistance phenotype in DX therapy sensitive cells by shRNA silencing of MFN2 or PACS2 and confirm their apoptotic resistance by BH3 profiling and drug response in vitro. A 60% decrease in MFN2 protein in DX cells partially phenocopied the resistance profile of isogenic REL cells. The degree of apoptotic resistance correlated with the extent of protein knockdown for MFN2. Treatment of shMFN2 and shPACS2 DX cells with ABT-737, a BH3 mimetic, or carboplatin, increased their IC50s multiple-fold compared to control cells, paralleling their blunted mitochondrial apoptotic response. Conclusions: Our data implicate ER-mito contact sites as positive regulators of apoptosis, whose disruption may be necessary for apoptotic attenuation in therapy resistant cancer cells to enable apoptosis evasion and survival. We present a potential mechanism for broad therapy resistance arising under therapeutic stress that selects for reduced communication of ER with mitochondria. Citation Format: Jorida Coku, Madison C. Pedrotty, David M. Booth, Sharon Kim, Annette Vu, C. Patrick Reynolds, György Hajnóczky, Michael D. Hogarty. Disrupted endoplasmic reticulum-mitochondria contacts promote multidrug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1201. doi:10.1158/1538-7445.AM2017-1201

A molecular genetic toolbox for Yarrowia lipolytica

BackgroundYarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.ResultsWe have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the “Yarrowia lipolytica Cell Atlas,” a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.ConclusionsThese molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.

Reactive Oxygen Species, Endoplasmic Reticulum Stress and Mitochondrial Dysfunction: The Link with Cardiac Arrhythmogenesis.

Background: Cardiac arrhythmias represent a significant problem globally, leading to cerebrovascular accidents, myocardial infarction, and sudden cardiac death. There is increasing evidence to suggest that increased oxidative stress from reactive oxygen species (ROS), which is elevated in conditions such as diabetes and hypertension, can lead to arrhythmogenesis.Method: A literature review was undertaken to screen for articles that investigated the effects of ROS on cardiac ion channel function, remodeling and arrhythmogenesis.Results: Prolonged endoplasmic reticulum stress is observed in heart failure, leading to increased production of ROS. Mitochondrial ROS, which is elevated in diabetes and hypertension, can stimulate its own production in a positive feedback loop, termed ROS-induced ROS release. Together with activation of mitochondrial inner membrane anion channels, it leads to mitochondrial depolarization. Abnormal function of these organelles can then activate downstream signaling pathways, ultimately culminating in altered function or expression of cardiac ion channels responsible for generating the cardiac action potential (AP). Vascular and cardiac endothelial cells become dysfunctional, leading to altered paracrine signaling to influence the electrophysiology of adjacent cardiomyocytes. All of these changes can in turn produce abnormalities in AP repolarization or conduction, thereby increasing likelihood of triggered activity and reentry.Conclusion: ROS plays a significant role in producing arrhythmic substrate. Therapeutic strategies targeting upstream events include production of a strong reducing environment or the use of pharmacological agents that target organelle-specific proteins and ion channels. These may relieve oxidative stress and in turn prevent arrhythmic complications in patients with diabetes, hypertension, and heart failure.

Abstract 2459: Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering

Abstract Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies. Citation Format: Jorida Coku, Elizabeth O. Scadden, Kangning Liu, Annette Vu, David M. Booth, Michelle Chen, Sharon Kim, C. Patrick Reynolds, György Hajnóczky, Michael D. Hogarty. Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2459.

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.

Interaction of the dual targeting peptide of Thr-tRNA synthetase with the chloroplastic receptor Toc34 in Arabidopsis thaliana

Organellar proteins synthesized in the cytosol are usually selective for only one destination in a cell but some proteins are localized in more than one compartment, for example in both mitochondria and chloroplasts. The mechanism of dual targeting of proteins to mitochondria and chloroplasts is yet poorly understood. Previously, we observed that the dual targeting peptide of threonyl-tRNA synthetase in Arabidopsis thaliana (AtThrRS-dTP) interacts with the mitochondrial receptor AtTom20 mainly through its N-terminal part. Here we report on the interaction of AtThrRS-dTP with the chloroplastic receptor AtToc34, presenting for the first time the mode of interactions of a dual targeting peptide with both Tom20 and Toc34. By NMR spectroscopy we investigated changes in 15N HSQC spectra of AtThrRS-dTP as a function of AtToc34 concentration. Line broadening shows that the interaction with AtToc34 involves residues along the entire sequence, which is not the case for AtTom20. The N-terminal φχχφφ motif, which plays an important role in AtTom20 recognition, shows no specificity for AtToc34. These results are supported by import competition studies into both mitochondria and chloroplasts, in which the effect of peptides corresponding to different segments of AtThrRS-dTP on in vitro import of organelle specific proteins was examined. This demonstrates that the N-terminal A2-Y29 segment of AtThrRS-dTP is essential for import into both organelles, while the C-terminal L30-P60 part is important for chloroplastic import efficiency. In conclusion, we have demonstrated that the recognition of the dual targeting peptide of AtThr-tRNA synthetase is different for the mitochondrial and chloroplastic receptors.

Experimental validation of predicted subcellular localizations of human proteins.

BackgroundComputational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations.FindingsWe have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines.ConclusionsThe experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-912) contains supplementary material, which is available to authorized users.

Cellular Communication Via Nanoparticle-Transporting Biovesicles

Endothelial cells are dynamic cells tasked with selective transport of cargo from blood vessels to tissues. Here we demonstrate the potential for nanoparticle transport across endothelial cells in membrane-bound vesicles. Cell-free endothelial-derived biovesicles were characterized for cellular and nanoparticle content by electron microscopy. Confocal microscopy was used to evaluate biovesicles for organelle-specific proteins, and to monitor biovesicle engulfment by naive cells. Nanoparticle-laden biovesicles containing low-density polyethyleneimine nanoparticles appear to be predominately of endosomal origin, combining features of multivesicular bodies, lysosomes and autophagosomes. Conversely, high-density polyethyleneimine nanoparticles stimulate the formation of biovesicles associated with cellular apoptotic breakdown. Secreted LAMP-1-positive biovesicles are internalized by recipient cells, either of the same origin or of novel phenotype. Cellular biovesicles, rich in cellular signals, present an important mode of cell-to-cell communication either locally or through broadcasting of biological messages.