Browsing by Subject "MEMBRANE"

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  • Dillard, Kati J.; Hytönen, Marjo K.; Fischer, Daniel; Tanhuanpää, Kimmo; Lehti, Mari S.; Vainio-Siukola, Katri; Sironen, Anu; Anttila, Marjukka (2018)
    Ciliopathies presenting as inherited hepatorenal fibrocystic disorders are rare in humans and in dogs. We describe here a novel lethal ciliopathy in Norwich Terrier puppies that was diagnosed at necropsy and characterized as diffuse cystic renal disease and hepatic fibrosis. The histopathological findings were typical for cystic renal dysplasia in which the cysts were located in the straight portion of the proximal tubule, and thin descending and ascending limbs of Henle's loop. The pedigree of the affected puppies was suggestive of an autosomal recessive inheritance and therefore, whole exome sequencing and homozygosity mapping were used for identification of the causative variant. The analyses revealed a case-specific homozygous splice donor site variant in a cilia related gene, INPP5E: c.1572+5G>A. Association of the variant with the defect was validated in a large cohort of Norwich Terriers with 3 cases and 480 controls, the carrier frequency being 6%. We observed that the identified variant introduces a novel splice site in INPP5E causing a frameshift and formation of a premature stop codon. In conclusion, our results suggest that the INPP5E: c.1572+5G>A variant is causal for the ciliopathy in Norwich Terriers. Therefore, genetic testing can be carried out in the future for the eradication of the disease from the breed.
  • Sola-Carvajal, Agustin; Revechon, Gwladys; Helgadottir, Hafdis T.; Whisenant, Daniel; Hagblom, Robin; Döhla, Julia; Katajisto, Pekka; Brodin, David; Fagerstrom-Billai, Fredrik; Viceconte, Nikenza; Eriksson, Maria (2019)
    Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/beta-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of beta-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.
  • Leopold, Anna; Pletnev, Sergei; Verkhusha, Vladislav V. (2020)
    Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLC gamma signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light. (C) 2020 Elsevier Ltd. All rights reserved.
  • Duyvesteyn, Helen M. E.; Santos-Pérez, Isaac; Peccati , Francesca; Martinez-Castillo, Ane; Walter, Thomas S.; Reguera , David; Goni, Felix M; Jiménez-Osés , Gonzalo; Oksanen, Hanna M; Stuart, David I.; Abrescia, Nicola GA (2021)
    Viruses are very attractive biomaterials owing to their capability as nanocarriers of genetic material. Efforts have been made to functionalize self-assembling viral protein capsids on their exterior or interior to selectively take up different payloads. PRD1 is a double-stranded DNA bacteriophage comprising an icosahedral protein outer capsid and an inner lipidic vesicle. Here, we report the three-dimensional structure of PRD1 in complex with the antipsychotic drug chlorpromazine (CPZ) by cryo-electron microscopy. We show that the jellyrolls of the viral major capsid protein P3, protruding outwards from the capsid shell, serve as scaffolds for loading heterocyclic CPZ molecules. Additional X-ray studies and molecular dynamics simulations show the binding modes and organization of CPZ molecules when complexed with P3 only and onto the virion surface. Collectively, we provide a proof of concept for the possible use of the lattice-like organisation and the quasi-symmetric morphology of virus capsomers for loading heterocyclic drugs with defined properties.
  • Dusa, Filip; Chen, Wen; Witos, Joanna; Wiedmer, Susanne Kristina (2019)
    The importance of using biomimicking membranes for various biological applications is rising, as such models are relevant for imitating real organisms. In addition, biomimicking membranes are usually much more repeatable in preparation and easier to handle during analysis than real organisms or biological membranes. In this work, we developed a method for the adsorption of intact small unilamellar Escherichia coli (E. cols) vesicles (Z-average size of 73 nm) on SiO2 substrate material. We describe the adsorption process based on the use of two surface sensitive techniques, i.e., nanoplasmonic sensing (NPS) and quartz crystal microbalance (QCM). The acquired data show that the adsorption follows a two-step process. The first step is a slow adsorption of E coil vesicle aggregates held together by 5 mM of calcium (Z-average size of 531 nm). The Z-average of the aggregates decreased almost three times when the calcium concentration was decreased to 0.1 mM. This suggests that the aggregates were disassembling to some extent when calcium was removed from the system. With both techniques, i.e., NPS and QCM, we observed a second rapid adsorption step after the solution was changed to deionized water. In this second step, the aggregates started to fall apart as the calcium concentration dropped, and the released vesicles started to adsorb onto unoccupied spots at the SiO2 surface of the sensors. Extensive release of mass from the surface was confirmed by QCM, where it was reflected by a sharp increase of frequency, while NPS, due to its lower sensing depth of a few tens of nanometers, did not record such a change. Taken together, we have developed a protocol to form a supported vesicle layer (SVL) of E coli vesicles on SiO2 surface using sodium 4-(2-hydroxyethyppiperazine-1-ethanesulfonate buffer, thus enabling the preparation of E coli biomimicking SVLs for interaction studies of compounds of interest. The immobilization happens via a two-step adsorption process.
  • Haapanen, Outi; Reidelbach, Marco; Sharma, Vivek (2020)
    Respiratory complex I (NADH:quinone oxidoreductase) plays a central role in generating the proton electrochemical gradient in mitochondrial and bacterial membranes, which is needed to generate ATP. Several high-resolution structures of complex I have been determined, revealing its intricate architecture and complementing the biochemical and biophysical studies. However, the molecular mechanism of long-range coupling between ubiquinone (Q) reduction and proton pumping is not known. Computer simulations have been applied to decipher the dynamics of Q molecule in the similar to 30 angstrom long Q tunnel. In this short report, we discuss the binding and dynamics of Q at computationally predicted Q binding sites, many of which are supported by structural data on complex I. We suggest that the binding of Q at these sites is coupled to proton pumping by means of conformational rearrangements in the conserved loops of core subunits.
  • Lauter, Gilbert; Coschiera, Andrea; Yoshihara, Masahito; Sugiaman-Trapman, Debora; Ezer, Sini; Sethurathinam, Shalini; Katayama, Shintaro; Kere, Juha; Swoboda, Peter (2020)
    Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing imecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.
  • Djurabekova, Amina; Yoga, Etienne Galemou; Nyman, Aino; Pirttikoski, Antti; Zickermann, Volker; Haapanen, Outi; Sharma, Vivek (2022)
    The first component of the mitochondrial electron transport chain is respiratory complex I. Several high-resolution structures of complex I from different species have been resolved. However, despite these significant achievements, the mechanism of redox-coupled proton pumping remains elusive. Here, we combined atomistic docking, molecular dynamics simulations, and site-directed mutagenesis on respiratory complex I from Yarrowia lipolytica to identify a quinone (Q)-binding site on its surface near the horizontal amphipathic helices of ND1 and NDUFS7 subunits. The surface-bound Q makes stable interactions with conserved charged and polar residues, including the highly conserved Arg72 from the NDUFS7 subunit. The binding and dynamics of a Q molecule at the surface-binding site raise interesting possibilities about the mechanism of complex I, which are discussed.
  • Grabon, Aby; Orlowski, Adam; Tripathi, Ashutosh; Vuorio, Joni; Javanainen, Matti; Rog, Tomasz; Lönnfors, Max; McDermott, Mark I.; Siebert, Garland; Somerharju, Pentti; Vattulainen, Ilpo; Bankaitis, Vytas A. (2017)
    Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITP alpha, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITP alpha lipid exchange cycle: (i) interaction of PITP alpha with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITP alpha to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITP alpha; (iv) the trajectory of PtdIns or PtdCho into and through the lipidbinding pocket is chaperoned by sets of PITP alpha residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITP alpha PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.
  • Morimoto, Nobuyuki; Takei, Riho; Wakamura, Masaru; Oishi, Yoshifumi; Nakayama, Masafumi; Suzuki, Makoto; Yamamoto, Masaya; Winnik, Francoise M. (2018)
    Mitochondrial targeting and entry, two crucial steps in fighting severe diseases resulting from mitochondria dysfunction, pose important challenges in current nanomedicine. Cell-penetrating peptides or targeting groups, such as Rhodamine-B (Rho), are known to localize in mitochondria, but little is known on how to enhance their effectiveness through structural properties of polymeric carriers. To address this issue, we prepared 8 copolymers of 3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate and poly(ethyleneglycol) methacrylate, p(DMAPS-ran-PEGMA) (molecular weight, 18.0 <M-n <74.0 kg/mol) with two different endgroups. We labeled them with Rho groups attached along the chain or on one of the two endgroups (alpha or omega). From studies by flow cytometry and confocal fluorescence microscopy of the copolymers internalization in HeLa cells in the absence and presence of pharmacological inhibitors, we established that the polymers cross the cell membrane foremost by translocation and also by endocytosis, primarily clathrin-dependent endocytosis. The most effective mitochondrial entry was achieved by copolymers of M-n <30.0 kg/mol, lightly grafted with PEG chains (<5 mol %) labeled with Rho in the omega-position. Our findings may be generalized to the uptake and mitochondrial targeting of prodrugs and imaging agents with a similar polymeric scaffold.
  • Gungor, Burcin; Vanharanta, Lauri; Hölttä-Vuori, Maarit; Pirhonen, Juho; Petersen, Nikolaj H. T.; Gramolelli, Silvia; Ojala, Päivi M.; Kirkegaard, Thomas; Ikonen, Elina (2019)
    Objective: Heat Shock Proteins (HSPs) maintain cellular homeostasis under stress. HSP70 represents a major stress-inducible family member and has been identified as a druggable target in inherited cholesterol-sphingolipid storage diseases. We investigated if HSP70 modulates cholesterol accumulation in more common conditions related to atherogenesis. Methods: We studied the effects of recombinant HSP70 in cholesterol-laden primary macrophages from human blood donors and pharmacological HSP70 upregulation in high-cholesterol diet fed zebrafish. Results: Recombinant HSP70 facilitated cholesterol removal from primary human macrophage foam cells. RNA sequencing revealed that HSP70 induced a robust transcriptional re-programming, including upregulation of key targets of liver X receptors (LXR), master regulators of whole-body cholesterol removal. Mechanistically, HSP70 interacted with the macrophage LXRalpha promoter, increased LXRalpha and its target mRNAs, and led to elevated levels of key proteins facilitating cholesterol efflux, including ATP-binding cassette transporters A1 and G1. Pharmacological augmentation of endogenous HSP70 in high-cholesterol diet fed zebrafish activated LXR and its target mRNAs and reduced cholesterol storage at the whole organism level. Conclusion: These data demonstrate that HSP70 exerts a cholesterol lowering effect in primary human cells and animals and uncover a nuclear action of HSP70 in mediating cross-talk between HSP and LXR transcriptional regulation. (C) 2019 The Authors. Published by Elsevier GmbH.
  • Poranen, Minna M.; Mäntynen, Sari; ICTV Report Consortium (2017)
    The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www. ictv. global/report/cystoviridae.
  • Parviainen, Suvi; Autio, Karoliina; Vähä-Koskela, Markus; Guse, Kilian; Pesonen, Sari; Rosol, Thomas J.; Zhao, Fang; Hemminki, Akseli (2015)
    Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.
  • Estonian Biobank Research Team; BioBank Japan Project; FinnGen; Ruotsalainen, Sanni E.; Surakka, Ida; Mars, Nina; Karjalainen, Juha; Kurki, Mitja; Kanai, Masahiro; Krebs, Kristi; Graham, Sarah; Mishra, Pashupati P.; Mishra, Binisha H.; Sinisalo, Juha; Palta, Priit; Lehtimäki, Terho; Raitakari, Olli; Milani, Lili; Okada, Yukinori; Palotie, Aarno; Widen, Elisabeth; Daly, Mark J.; Ripatti, Samuli (2022)
    A genome-wide association study identifies MFGE8 as protective against coronary atherosclerosis in European and East Asian populations. Cardiovascular diseases are the leading cause of premature death and disability worldwide, with both genetic and environmental determinants. While genome-wide association studies have identified multiple genetic loci associated with cardiovascular diseases, exact genes driving these associations remain mostly uncovered. Due to Finland's population history, many deleterious and high-impact variants are enriched in the Finnish population giving a possibility to find genetic associations for protein-truncating variants that likely tie the association to a gene and that would not be detected elsewhere. In a large Finnish biobank study FinnGen, we identified an association between an inframe insertion rs534125149 in MFGE8 (encoding lactadherin) and protection against coronary atherosclerosis. This variant is highly enriched in Finland, and the protective association was replicated in meta-analysis of BioBank Japan and Estonian biobank. Additionally, we identified a protective association between splice acceptor variant rs201988637 in MFGE8 and coronary atherosclerosis, independent of the rs534125149, with no significant risk-increasing associations. This variant was also associated with lower pulse pressure, pointing towards a function of MFGE8 in arterial aging also in humans in addition to previous evidence in mice. In conclusion, our results suggest that inhibiting the production of lactadherin could lower the risk for coronary heart disease substantially.
  • Heinonen, Suvi; Lautala, Saara; Koivuniemi, Artturi; Bunker, Alex (2022)
    The lipid second messenger diacylglycerol (DAG) is known for its involvement in many types of cellular signaling, especially as an endogenous agonist for protein kinase C (PKC). Evidence has emerged that the degree of saturation of the DAG molecules can affect PKC activation. DAG molecules with different acyl chain saturation have not only been observed to induce varying extents of PKC activation, but also to express selectivity towards different PKC isozymes. Both qualities are important for precise therapeutic activation of PKC; understanding DAG behavior at the molecular level in different environments has much potential in the development of drugs to target PKC. We used molecular dynamics simulations to study the behavior of two different unsaturated DAG species in lipid environments with varying degrees of unsaturation. We focus on phosphatidylethanolamine (PE) instead of phosphatidylcholine (PC) to more accurately model the relevant biomembranes. The effect of cholesterol (CHOL) on these systems was also explored. We found that both high level of unsaturation in the acyl chains of the DAG species and presence of CHOL in the surrounding membrane increase DAG molecule availability at the lipid–water interface. This can partially explain the previously observed differences in PKC activation strength and specificity, the complete mechanism is, however, likely to be more complex. Our simulations coupled with the current understanding of lipids highlight the need for more simulations of biologically accurate lipid environments in order to determine the correct correlations between molecular mechanisms and biological behavior when studying PKC activation.
  • Senju, Yosuke; Lappalainen, Pekka; Zhao, Hongxia (Humana press, 2021)
    Methods in Molecular Biology
    A large proportion of proteins are expected to interact with cellular membranes to carry out their physiological functions in processes such as membrane transport, morphogenesis, cytoskeletal organization, and signal transduction. The recruitment of proteins at the membrane-cytoplasm interface and their activities are precisely regulated by phosphoinositides, which are negatively charged phospholipids found on the cytoplasmic leaflet of cellular membranes and play critical roles in membrane homeostasis and cellular signaling. Thus, it is important to reveal which proteins interact with phosphoinositides and to elucidate the underlying mechanisms. Here, we present two standard in vitro methods, liposome co-sedimentation and co-flotation assays, to study lipid-protein interactions. Liposomes can mimic various biological membranes in these assays because their lipid compositions and concentrations can be varied. Thus, in addition to mechanisms of lipid-protein interactions, these methods provide information on the possible specificities of proteins toward certain lipids such as specific phosphoinositide species and can hence shed light on the roles of membrane interactions on the functions of membrane-associated proteins.
  • Salmenkari, Hanne; Issakainen, Tomi; Vapaatalo, Heikki; Korpela, Riitta (2015)
    AIM: To investigate local corticosterone production and angiotensin- I converting enzyme (ACE) protein expression and their interaction in healthy and inflamed intestine. METHODS: Acute intestinal inflammation was induced to six weeks old male Balb/c mice by administration of either 3% or 5% dextran sodium sulfate (DSS) in drinking water for 7 d (n = 12 in each group). Healthy controls (n = 12) were given tap water. Corticosterone production and ACE protein shedding were measured from ex vivo incubates of the small and large intestine using EIA and ELISA, respectively. Morphological changes of the intestinal wall were assessed in hematoxylin-eosin stained tissue preparations of jejunum and distal colon. Effects of angiotensin II, captopril and metyrapone on corticosterone production was assessed by incubating pieces of small intestine of healthy mice in the presence of 0.1, 1 or 10 mu mol/L angiotensin II, 1, 10 or 100 mu mol/L captopril or 1, 10 or 100 mu mol/L metyrapone solutions and measuring corticosterone released to the incubation buffer after 90 min (n = 5 in each group). RESULTS: Both concentrations of DSS induced inflammation and morphological changes in large intestines but not in small intestines. Changes were observed as distortions of the crypt structure, mucosal erosion, immune cell infiltration to the mucosa and submucosal edema. Ex vivo corticosterone production (2.9 +/- 1.0 ng/mL vs 2.0 +/- 0.8 ng/mL, P = 0.034) and ACE shedding (269.2 +/- 97.1 ng/mL vs 175.7 +/- 52.2 ng/mL, P = 0.016) were increased in small intestines in 3% DSS group compared to the controls. In large intestine, corticosterone production was increased compared to the controls in both 3% DSS (229 +/- 81 pg/mL vs 158 +/- 30 pg/mL, P = 0.017) and 5% DSS groups (366 +/- 163 pg/mL vs 158 +/- 30 pg/mL, P = 0.002). Large intestine ACE shedding was increased in 5% DSS group (41.5 +/- 9.0 ng/mL vs 20.9 +/- 5.2 ng/mL, P = 0.034). Angiotensin II treatment augmented corticosterone production in small intestine at concentration of 10 mu mol/L (0.97 +/- 0.21 ng/mg protein vs 0.40 +/- 0.09 ng/mg protein, P = 0.036). CONCLUSION: Intestinal ACE shedding is increased by DSS-induced intestinal inflammation and parallels local corticosterone production. ACE product angiotensin. stimulates corticosterone formation in healthy intestine.
  • Lyytinen, Outi Leena; Starkova, Daria; Poranen, Minna Marjetta (2019)
    BackgroundCystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli.ResultsUsing transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13g/cm(3) in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles.ConclusionsOur results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.
  • Richter, Uwe; Ng, Kah Ying; Suomi, Fumi; Marttinen, Paula; Turunen, Taina; Jackson, Christopher; Suomalainen, Anu; Vihinen, Helena; Jokitalo, Eija; Nyman, Tuula A.; Isokallio, Marita A.; Stewart, James B.; Mancini, Cecilia; Brusco, Alfredo; Seneca, Sara; Lombes, Anne; Taylor, Robert W.; Battersby, Brendan J. (2019)
    Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.
  • Gebraad, Arjen; Kornilov, Roman; Kaur, Sippy; Miettinen, Susanna; Haimi, Suvi; Peltoniemi, Hilkka; Mannerström, Bettina; Seppänen-Kaijansinkko, Riitta (2018)
    Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. DatabaseGene expression data are available in the GEO database under the accession number .