Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli

F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fimbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specific promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specific systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specific immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were significantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The findings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.

Multifunctional RNA silencing pathway polymerase QDE-1 of Neurospora crassa

Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms.

Functional characterization of MutL homologue mismatch repair proteins and their variants

Mismatch repair (MMR) mechanisms repair DNA damage occurring during replication and recombination. To date, five human MMR genes, MSH2, MHS6, MSH3, MLH1 and PMS2 are known to be involved in the MMR function. Human MMR proteins form 3 different heterodimers: MutSα (MSH2 and MSH6) and MutSβ (MSH2 and MSH3), which are needed for mismatch recognition and binding, and MutLα (MLH1 and PMS2), which is needed for mediating interactions between MutS homologues and other MMR proteins. The other two MutL homologues, MLH3 and PMS1, have been shown to heterodimerize with MLH1. However, the heterodimers MutLγ (MLH1and MLH3) and MutLβ (MLH1 and PMS1) are able to correct mismatches only with low or no efficiency, respectively. A deficient MMR mechanism is associated with the hereditary colorectal cancer syndrome called hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. HNPCC is the most common hereditary colorectal cancer syndrome and accounts for 2-5% of all colorectal cancer cases. HNPCC-associated mutations have been found in 5 MMR genes: MLH1, MSH2, MSH6, PMS2 and MLH3. Most of the mutations have been found in MLH1 and MSH2 (~90%) and are associated with typical HNPCC, while mutations in MSH6, PMS2 and MLH3 are mainly linked to putative HNPCC families lacking the characteristics of the syndrome. More data of MLH3 mutations are needed to assess the significance of its mutations in HNPCC. In this study, were functionally characterized 51 nontruncating mutations in the MLH1, MLH3 and MSH2 genes to address their pathogenic significance and mechanism of pathogenicity. Of the 36 MLH1 mutations, 22 were deficient in more than one assay, 2 variants were impaired only in one assay, and 12 variants behaved like the wild type protein, whereas all seven MLH3 mutants functioned like the wild type protein in the assays. To further clarify the role and relevance of MLH3 in MMR, we analyzed the subcellular localization of the native MutL homologue proteins. Our immunofluorescence analyses indicated that when all the three MutL homologues are natively expressed in human cells, endogenous MLH1 and PMS2 localize in the nucleus, whereas MLH3 stays in the cytoplasm. The coexpression of MLH3 with MLH1 results in its partial nuclear localization. Only one MSH2 mutation was pathogenic in the in vitro MMR assay. Our study on MLH1 mutations could clearly distinguish nontruncating alterations with severe functional defects from those not or only slightly impaired in protein function. However, our study on MLH3 mutations suggest that MLH3 mutations per se are not sufficient to trigger MMR deficiency and the continuous nuclear localization of MLH1 and PMS2 suggest that MutLα has a major activity in MMR in vivo. Together with our functional assays, this confirms that MutLγ is a less efficient MMR complex than MutLα.

Prevalence and analysis of t(14;18) and t(11;14) chromosomal translocations in healthy Indian population

Hematopoietic malignancies like leukemia and lymphoma are characteristically associated with various chromosomal translocations. Follicular lymphoma (FL) and mantle cell lymphoma (MCL) are two subtypes of non-Hodgkin's lymphoma which possess t(14;18) and t(11;14) translocations, respectively. The incidence of FL and MCL is higher in the western countries as compared to India. Interestingly, the associated translocations are also found in healthy individuals in western population, which is 50-80% for t(14;18), whereas t(11;14) occurs at a very low frequency. However, there are no studies to explore thes translocations in healthy Indian population, which could explain the lower incidence of FL and MCL. We employed Southern hybridization following nested PCR to detect above translocations in healthy individuals from India. Our results suggest that this assay can detect one t(14;18) translocation event in up to 10(7) normal cells where as one t(11;14) in 10(8) normal cells. According to our results, 87 out of 253 individuals carry t(14;18) indicating 34% prevalence in the population. The presence of this translocation was also detectable at the transcript level. Although, no gender-based difference was observed, an age-dependent increase in the prevalence of translocation was found in adults. However, even after studying 210 people, we could not detect any t(11;14) translocation, indicating that it is uncommon in Indian population. These results suggest that lower incidence of FL and MCL in India could be attributed to lower prevalence of these translocations in healthy individuals.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling

Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor-kappa B (NF-kappa B) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of uppressor of Hairless (CSL) and NF-kappa B to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli.

The type III secretion system (T3SS) encoded by the Salmonella pathogenicity island 2 (SPI2) has a central role in systemic infections by Salmonella enterica and for the intracellular phenotype. Intracellular S. enterica uses the SPI2-encoded T3SS to translocate a set of effector proteins into the host cell, which modify host cell functions, enabling intracellular survival and replication of the bacteria. We sought to determine whether specific functions of the SPI2-encoded T3SS can be transferred to heterologous hosts Salmonella bongori and Escherichia coli Mutaflor, species that lack the SPI2 locus and loci encoding effector proteins. The SPI2 virulence locus was cloned and functionally expressed in S. bongori and E. coli. Here, we demonstrate that S. bongori harboring the SPI2 locus is capable of secretion of SPI2 substrate proteins under culture conditions, as well as of translocation of effector proteins under intracellular conditions. An SPI2-mediated cellular phenotype was induced by S. bongori harboring the SPI2 if the sifA locus was cotransferred. An interference with the host cell microtubule cytoskeleton, a novel SPI2-dependent phenotype, was observed in epithelial cells infected with S. bongori harboring SPI2 without additional effector genes. S. bongori harboring SPI2 showed increased intracellular persistence in a cell culture model, but SPI2 transfer was not sufficient to confer to S. bongori systemic pathogenicity in a murine model. Transfer of SPI2 to heterologous hosts offers a new tool for the study of SPI2 functions and the phenotypes of individual effectors.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Identification of novel target genes in different subtypes of cutaneous T-cell lymphoma

Ihon T-solulymfoomat (cutaneous T-cell lymphoma, CTCL) ovat ryhmä imukudossyöpiä, joiden esiintyvyys on nousussa erityisesti länsimaissa. Taudin syntymekanismit ovat suurelta osin tuntemattomat, diagnostiikka on vaikeaa ja siksi usein viivästynyttä eikä parantavaa hoitoa ole. CTCL ilmenee iho-oirein, vaikka syöpäsolut eivät ole iholla normaalisti esiintyviä soluja, vaan elimistön puolustusjärjestelmän soluja, jotka ovat tuntemattomasta syystä vaeltaneet iholle. Syöpäsolut ovat kypsiä T-auttajasoluja (Th-soluja) ja ilmentävät tyypin 2 immuunivasteelle ominaisia sytokiineja. Kromosomaalinen epästabiilius on tautiryhmän keskeinen piirre. CTCL-potilailla on lisääntynyt riski sairastua myös muihin syöpiin, erityisesti keuhkosyöpään ja non-Hodgkin –lymfoomiin. Väitöskirjatutkimuksen tavoitteena oli havaita CTCL:n syntymekanismeja selvittäviä kromosomi- ja geenimuutoksia. Erityisesti tavoitteena oli identifioida molekyylejä, jotka soveltuisivat diagnostisiksi merkkiaineiksi tai täsmähoidon kohteeksi. Työssä on tutkittu kahta tautiryhmän yleisintä muotoa, mycosis fungoidesta (MF) ja Sezaryn syndroomaa (SS) sekä harvinaisempaa vaikeasti diagnosoitavaa subkutaanista pannikuliitin kaltaista T-solulymfoomaa (SPTL). Lisäksi on tutkittu CTCL:ään liittyvää keuhkosyöpää ja verrattu sitä tavalliseen (primaariin) keuhkosyöpään. Tutkimusmenetelminä on käytetty esimerkiksi molekyylisytogeneettisiä metodeja ja mikrosiruja. Väitöskirjatyössä havaittiin ensimmäinen CTCL:lle ominainen toistuva geenitason muutos: puutos- tai katkoskohta NAV3-geenissä. Tämän geenipoikkeavuuden havaittiin esiintyvän useissa taudin alaryhmissä (MF, SS, SPTL). NAV3-geenipuutoksen osoittaminen FISH-tekniikalla on sovellettavissa kliiniseen diagnostiikkaan. Tutkimukset geenipuutoksen aiheuttamista toiminnallisista seurauksista ovat käynnissä. Työssä saatiin myös uutta tietoa taudin syntymekanismeista havaitsemalla useiden Th1-tyypin immuunivasteelle ominaisten geenien alentunut ilmeneminen CTCL-potilailla. Tämän lisäksi potilasnäytteissä havaittiin eräiden solun pinta-antigeenien lisääntynyt ilmeneminen, mikä luo pohjan uusien vasta-ainepohjaisten täsmähoitojen kehittämiselle. Väitöskirjatutkimuksessa todettiin myös CTCL:ään liittyvän keuhkosyövän eroavan kromosomi- ja geenimuutosten suhteen verrokkikeuhkosyövästä, mikä jatkossa antaa aiheen tutkia syöpäkantasolujen merkitystä CTCL:n ja sen liitännäiskasvainten kehittymisen taustalla.

Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha

We have overexpressed an 8.5-kDa mouse Ca2+/calmodulin kinase II inhibitor a protein (mCaMKIIN alpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca2+/calmodulin kinase 11 (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIIN alpha. react with an similar to 37-kDa protein present in mouse brain. The pattern of expression of the similar to 37-kDa protein is similar to that of mCaMKIIN alpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the similar to 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKII alpha is known to perform key regulatory functions. We conclude that the similar to 37-kDa protein identified in this study is mCaMKIIN alpha. and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKII alpha. (c) 2007 Elsevier B.V. All rights reserved.

Characterization of LRRTM and NGR gene families: Expression and functions

Some leucine-rich repeat (LRR) -containing membrane proteins are known regulators of neuronal growth and synapse formation. In this work I characterize two gene families encoding neuronal LRR membrane proteins, namely the LRRTM (leucine-rich repeat, transmembrane neuronal) and NGR (Nogo-66 receptor) families. I studied LRRTM and NGR family member's mRNA tissue distribution by RT-PCR and by in situ hybridization. Subcellular localization of LRRTM1 protein was studied in neurons and in non-neuronal cells. I discovered that LRRTM and NGR family mRNAs are predominantly expressed in the nervous system, and that each gene possesses a specific expression pattern. I also established that LRRTM and NGR family mRNAs are expressed by neurons, and not by glial cells. Within neurons, LRRTM1 protein is not transported to the plasma membrane; rather it localizes to endoplasmic reticulum. Nogo-A (RTN4), MAG, and OMgp are myelin-associated proteins that bind to NgR1 to limit axonal regeneration after central nervous system injury. To better understand the functions of NgR2 and NgR3, and to explore the possible redundancy in the signaling of myelin inhibitors of neurite growth, I mapped the interactions between NgR family and the known and candidate NgR1 ligands. I identified high-affinity interactions between RTN2-66, RTN3-66 and NgR1. I also demonstrate that Rtn3 mRNA is expressed in the same glial cell population of mouse spinal cord white matter as Nogo-A mRNA, and thus it could have a role in myelin inhibition of axonal growth. To understand how NgR1 interacts with multiple structurally divergent ligands, I aimed first to map in more detail the nature of Nogo-A:NgR1 interactions, and then to systematically map the binding sites of multiple myelin ligands in NgR1 by using a library of NgR1 expression constructs encoding proteins with one or multiple surface residues mutated to alanine. My analysis of the Nogo-A:NgR1 -interactions revealed a novel interaction site between the proteins, suggesting a trivalent Nogo-A:NgR1-interaction. Our analysis also defined a central binding region on the concave side of NgR1's LRR domain that is required for the binding of all known ligands, and a surrounding region critical for binding MAG and OMgp. To better understand the biological role of LRRTMs, I generated Lrrtm1 and Lrrtm3 knock out mice. I show here that reporter genes expressed from the targeted loci can be used for maping the neuronal connections of Lrrtm1 and Lrrtm3 expressing neurons in finer detail. With regard to LRRTM1's role in humans, we found a strong association between a 70 kb-spanning haplotype in the proposed promoter region of LRRTM1 gene and two possibly related phenotypes: left-handedness and schizophrenia. Interestingly, the responsible haplotype was linked to phenotypic variability only when paternally inherited. In summary, I identified two families of neuronal receptor-like proteins, and mapped their expression and certain protein-protein interactions. The identification of a central binding region in NgR1 shared by multiple ligands may facilitate the design and development of small molecule therapeutics blocking binding of all NgR1 ligands. Additionally, the genetic association data suggests that allelic variation upstream of LRRTM1 may play a role in the development of left-right brain asymmetry in humans. Lrrtm1 and Lrrtm3 knock out mice developed as a part of this study will likely be useful for schizophrenia and Alzheimer s disease research.

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of Delta C-p of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of delta pam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

USING FLUORESCENT AND NON-FLUORESCENT STEROLS TO STUDY RAFTS AND INTRACELLULAR CHOLESTEROL TRANSPORT IN MAMMALIAN CELLS

Cholesterol is an essential component in the membranes of most eukaryotic cells, in which it mediates many functions including membrane fluidity, permeability and the formation of ordered membrane domains. In this work a fluorescent and a non-fluorescent cholesterol analog were characterized as tools to study cholesterol. Next, these analogs were used to study two specific cell biological processes that involve cholesterol, i.e. the structure and function of ordered membrane domains/rafts and intracellular cholesterol transport. The most common method for studying ordered membrane domains is by disrupting them by cholesterol depletion. Because cholesterol depletion affects many cellular functions besides those mediated by membrane domains, this procedure is highly unspecific. The cellular exchange of cholesterol by desmosterol as a tool to study ordered membrane domains was characterized. It turned out that the ability of desmosterol to form and stabilize membrane domains in vitro was weaker compared to cholesterol. This result was reinforced by atomistic scale simulations that indicated that desmosterol has a lower ordering effect on phospholipid acyl chains. Three procedures were established for exchanging cellular cholesterol by desmosterol. In cells in which desmosterol was the main sterol, insulin signaling was attenuated. The results suggest that this was caused by desmosterol destabilizing membrane rafts. Contrary to its effect on ordered membrane domains it was found that replacing cholesterol by desmosterol does not change cell growth/viability, subcellular sterol distribution, Golgi integrity, secretory pathway, phospholipid composition and membrane fluidity. Together these results suggest that exchanging cellular cholesterol by desmosterol provides a selective tool for perturbing rafts. Next, the importance of cholesterol for the structure and function of caveolae was analyzed by exchanging the cellular cholesterol by desmosterol. The sterol exchange reduced the stability of caveolae as determined by detergent resistance of caveolin-1 and heat resistance of caveolin-1 oligomers. Also the sterol exchange led to aberrations in the caveolar structure; the morphology of caveolae was altered and there was a larger variation in the amount of caveolin-1 molecules per caveola. These results demonstrate that cholesterol is important for caveolar stability and structural homogeneity. In the second part of this work a fluorescent cholesterol analog was characterized as a tool to study cholesterol transport. Tight control of the intracellular cholesterol distribution is essential for many cellular processes. An important mechanism by which cells regulate their membrane cholesterol content is by cholesterol traffic, mostly from the plasma membrane to lipid droplets. The fluorescent sterol probe BODIPY-cholesterol was characterized as a tool to analyze cholesterol transport between the plasma membrane, the endoplasmic reticulum (ER) and lipid droplets. The behavior of BODIPY-cholesterol was compared to that of natural sterols, using both biochemical and live-cell microcopy assays. The results show that the transport kinetics of BODIPY-cholesterol between the plasma membrane, the ER and lipid droplets is similar to that of unesterified cholesterol. Next, BODIPY-cholesterol was utilized to analyze the importance of oxysterol binding protein related proteins (ORPs) for cholesterol transport between the plasma membrane, the ER, and lipid droplets in mammalian cells. By overexpressing all human ORPs it turned out that especially ORP1S and ORP2 enhanced sterol transport from the plasma membrane to lipid droplets. Our results suggest that the increased sterol transport takes place between the plasma membrane and ER and not between the ER and lipid droplets. Simultaneous knockdown of ORP1S and ORP2 resulted in a moderate but significant inhibition of sterol traffic from the plasma membrane to ER and lipid droplets, suggesting a physiological role for these ORPs in this process. The two phenylalanines in an acidic tract (FFAT) motif in ORPs, which mediates interaction with vesicle associated membrane protein associated proteins (VAPs) in the ER, was not necessary for mediating sterol transport. However, VAP silencing slowed down sterol transport, most likely by destabilizing ORPs containing a FFAT motif.

Biochemical Effects of the Porphyrinogenic Drug Allyisopropylacetamide

Successive administrations of allylisopropylacetamide, a potent porphyrinogenic drug, increase liver weight, microsomal protein and phospholipid contents. There is an increase in the rate of microsomal protein synthesis in vivo and in vitro. The drug decreases microsomal ribonuclease activity and increases NADPH-cytochrome c reductase activity. Phenobarbital, which has been reported to exhibit all these changes mentioned, is a weaker inducer of delta-aminolaevulinate synthetase and increases the rate of haem synthesis only after a considerable time-lag in fed female rats, when compared with the effects observed with allylisopropylacetamide. Again, phenobarbital does not share the property of allylisopropylacetamide in causing an initial decrease in cytochrome P-450 content. Haematin does not counteract most of the biochemical effects caused by allylisopropylacetamide, although it is quite effective in the case of phenobarbital. Haematin does not inhibit the uptake of [2-(14)C]allylisopropylacetamide by any of the liver subcellular fractions.