bluuebunny/crossref_metadata_2025_split huggingface dataset:
A submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells
Authors: Schmidt Katja , Dartsch Heidrun , Linder Dietmar , Kern Horst-Franz , Kleene Ralf 
Published: April 2021

ABSTRACT
The secretory lectin ZG16p mediated the binding of aggregated zymogens to the granule membrane in pancreatic acinar cells. Using a recently established in vitro condensation-sorting assay, we now show that pretreatment of zymogen granule membranes (ZGM) with either sodium bicarbonate at pH 10 or with phosphatidyl inositol-specific phospholipase C (PI-PLC) reduced the binding efficiency of zymogens to the same extent, as distinct components were liberated from ZGM. Analysis of the composition of the bicarbonate extract revealed the presence of the secretory lectin ZG16p, the serpin ZG46p and the GPI-linked glycoprotein GP-2, together with several unknown proteins, and small amounts of lipase and carboxylester lipase. The unknown proteins detected in 2-D gels represented a group of acidic and basic protein spots, which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and several of the basic group reacted with a monoclonal antibody directed against chondroitin sulfate, indicating that the proteins represented proteoglycans. A staining pattern similar to the glycan reaction was observed in immunoblots using a polyclonal antibody directed against the whole bicarbonate extract. Immunogold electron microscopy revealed that this antibody reacted with components in the periphery of zymogen granules and strongly stained ZGM in the pellet fraction of a standard in vitro condensation-sorting assay. The amino acid composition of isolated components of both the acidic and basic group showed similarities to aggrecan, a cartilage-specific proteoglycan, and to glycine-rich glycoproteins, respectively. We therefore conclude that a submembranous matrix on the ZGM composed of proteoglycans and glycoproteins is involved in granule formation in pancreatic acinar cells.
An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata
Authors: Shiels Brian , Fox Mark , McKellar Sue , Kinnaird Jane , Swan David 
Published: April 2021

ABSTRACT
Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5′ upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5′-TTTGTAGGG-3′). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.
The central matrix loop drives import of uncoupling protein 1 into mitochondria
Authors: Schleiff Enrico , McBride Heidi 
Published: April 2021

ABSTRACT
The uncoupling protein (UCP1) is a carrier protein of the inner mitochondrial membrane spanning the bilayer six times. It does not contain a typical amino-terminal targeting signal and the mechanism of targeting and insertion is unknown. Here we focus on the biogenesis of UCP1 by analysing the import signals contained within the three repeated units of the protein. The amino-terminal third of the protein can mediate insertion into the outer membrane and therefore acts as artificial targeting signal when fused to DHFR. However, in the context of full-length UCP, the targeting information contained within the first repeated unit is not sufficient to trigger insertion into the outer membrane. Deletion of either the first or third repeated unit from UCP1 did not reduce import into the inner membrane and bound to the outer membrane receptor protein hTom20 with the characteristics of full-length UCP1. Deletion of the second repeat of UCP1 completely abolished all import into the mitochondria. Consistent with this, the central repeat alone was efficiently imported to the inner membrane and bound hTom20 with the characteristics of UCP1. We conclude that the site for binding hTom20 is within the central repeat and that this domain contains the complete targeting signal for directing UCP1 to the inner membrane.
Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA
Authors: Calado Angelo , Carmo-Fonseca Maria 
Published: April 2021

ABSTRACT
The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.
Roles of phospholipid signaling in chemoattractant-induced responses
Authors: Wu Dianqing , Huang Chi-Kuang , Jiang Huiping 
Published: April 2021

ABSTRACT
Chemoattractants, including chemokines, play a central role in regulation of inflammatory reactions by attracting and activating leukocytes. These molecules have been found to regulate metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) via phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Recent studies of mouse lines that lack PLC-β2, PLC-β3, or PI3Kγ demonstrate that chemoattractants act through PLC-β2 and PLC-β3 to hydrolyze PtdIns(4,5)P2 and through PI3Kγ to phosphorylate PtdIns(4,5)P2 in mouse neutrophils. These studies also confirmed the importance and revealed new roles of these signaling pathways in chemoattractant-induced responses.
The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor
Authors: Poukka Hetti , Karvonen Ulla , Yoshikawa Noritada , Tanaka Hirotoshi , Palvimo Jorma J. , Jänne Olli A. 
Published: April 2021

ABSTRACT
The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (≥90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (&lt;40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised (≤20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen (≤20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions.
Activation of protein kinase Cα inhibits growth of pancreatic cancer cells via p21cip-mediated G1 arrest
Authors: Detjen K. M. , Brembeck F. H. , Welzel M. , Kaiser A. , Haller H. , Wiedenmann B. , Rosewicz S. 
Published: April 2021

ABSTRACT
We have analyzed human pancreatic cancer cells to explore the growth regulatory function of protein kinase C (PKC)α. PKCα subcellular redistribution, activation kinetics and downregulation were examined in detail and correlated to immediate and delayed effects on cell-cycle regulatory pathways. TPA treatment resulted in transient PKCα activation accompanied by translocation of the enzyme into membrane and nuclear compartments, and was followed by subsequent downregulation. TPA-induced inhibition of DNA synthesis was prevented by a PKC-antagonist and was reproduced by microinjection of recombinant PKCα, indicating that activation of this isoenzyme was required and sufficient for growth inhibitory effects. PKCα activation arrested cells in the G1 phase of the cell cycle as a consequence of selective inhibition of cyclin dependent kinase (CDK)2 activity with concomitant hypophosphorylation of Rb. The inhibition of CDK2 activity resulted from induction of p21cip1 cyclin-dependent kinase inhibitors. Levels of p21cip1 remained elevated and CDK2 activity repressed in spite of PKCα downregulation, indicating that downstream effectors of PKCα are the primary determinants for the duration of PKC-mediated growth inhibition. The PKCα-induced block in cell proliferation persisted even though cells were kept in the presence of growth factors, suggesting that induction of PKCα results in a permanent withdrawal of pancreatic cancer cells from the cell cycle.
Pyk2 and FAK differentially regulate progression of the cell cycle
Authors: Zhao Jihe , Zhen Chuanhai , Guan Jun-Lin 
Published: April 2021

ABSTRACT
We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G1 to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant ΔC14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.
Drosophila D-Titin is required for myoblast fusion and skeletal muscle striation
Authors: Zhang Yong , Featherstone David , Davis Warren , Rushton Emma , Broadie Kendal 
Published: April 2021

ABSTRACT
An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains.
Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.
Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network
Authors: Blum Robert , Stephens David J. , Schulz Irene 
Published: April 2021

ABSTRACT
The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 μm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo.
Movies available on-line (http://www.biologists.com/JCS/movies/jcs1334.html), Movie ADDITION, Movie FIGURE 2, Movie FIGURE 3, Movie FIGURE 4, Movie FRAP, Movie GFP_P23, Movie OVERVIEW, Movie POST_GOL
Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of α5β1 integrin expression
Authors: Duncan Melinda K. , Kozmik Zbynek , Cveklova Kveta , Piatigorsky Joram , Cvekl Ales 
Published: April 2021

ABSTRACT
The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120ctn as well as large aggregates of α5β1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human α5- and β1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human α5 and β1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.
The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1
Authors: Johansson Ann-Sofi , Driessens Mariette , Aspenström Pontus 
Published: April 2021

ABSTRACT
A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42. PAR-6 contains a PDZ domain and in C. elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo. Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays. Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6. We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus. Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown. In C. elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex. In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus. Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex. These findings, together with the reported roles of PAR-6 and PAR-3 in C. elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.
Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex
Authors: Pistor Susanne , Gröbe Lothar , Sechi Antonio S. , Domann Eugen , Gerstel Birgit , Machesky Laura M. , Chakraborty Trinad , Wehland Jürgen 
Published: April 2021

ABSTRACT
The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.
The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL
Authors: Dragonetti Antonella , Baldassarre Massimiliano , Castino Roberta , Démoz Marina , Luini Alberto , Buccione Roberto , Isidoro Ciro 
Published: April 2021

ABSTRACT
Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-membrane receptors. The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophilic leukemia (RBL)/mast cell line to verify the hypothesis that inflammatory granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysosomal enzymes and are accessible by fluid-phase and membrane endocytosis markers. In addition, we studied the targeting, subcellular localisation and regulated secretion of the lysosomal aspartic protease cathepsin D (CD) as affected by IgE receptor stimulation in order to obtain information on the pathways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of specific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be prevented by addition of EGTA, indicating that it was dependent on extracellular calcium influx. Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an intermediate station in the pathway for granule biogenesis still sensitive to regulated exocytosis. The targeting of molecules to secretory granules may occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-independent pathway. We conclude that endosomes and lysosomes in basophils/mast cells can act as regulated secretory granules or actually identify with them.