scholarly journals Two novel phosphatidylinositol-4-phosphate 5-kinase type Iγ splice variants expressed in human cells display distinctive cellular targeting

2009 ◽  
Vol 422 (3) ◽  
pp. 473-482 ◽  
Author(s):  
Nicholas J. Schill ◽  
Richard A. Anderson
Keyword(s):  
Amino Acids ◽  
Splice Variant ◽  
Protein Targeting ◽  
Splice Variants ◽  
Human Cells ◽  
Subcellular Targeting ◽  
Splice Isoforms ◽  
Messenger Molecules ◽  
Phosphatidylinositol 4

The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger PtdIns(4,5)P2 is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type Iγ isoform of PIPK (PtdIns4P 5-kinase), which generate PtdIns(4,5)P2, have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKIγ in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in multiple tissue types and display PIPK activity in vitro. Interestingly, both of these novel splice variants display distinct subcellular targeting. With the addition of these two new splice isoforms, there are minimally five PIPKIγ splice variants that have been identified in mammals. Therefore, we propose the use of the HUGO (Human Genome Organization) nomenclature in the naming of the splice isoforms. PIPKIγ_i4 (700 amino acids) is present in the nucleus, a targeting pattern that has not been previously observed in any PIPKIγ splice variant. PIPKIγ_i5 (707 amino acids) is targeted to intracellular vesicle-like structures, where it co-localizes with markers of several types of endosomal compartments. As occurs with other PIPKIγ splice variants, the distinctive C-terminal sequences of PIPKIγ_i4 and PIPKIγ_i5 may facilitate association with unique protein targeting factors, thereby localizing the kinases to their appropriate cellular subdomains for the site-specific generation of PtdIns(4,5)P2.

scholarly journals Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Sara L. Seegers ◽  
Amanda Lance ◽  
Lawrence J Druhan ◽  
Belinda R Avalos
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Human Cells ◽  
Dependent Manner ◽  
Epitope Tag ◽  
Proliferative Signaling ◽  
Primary Human Cells

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

Characterization of Dendritic Cell-Specific Genes p275 and p306.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3842-3842
Author(s):  
Ingo Hilgendorf ◽  
Daniel Kurz ◽  
Anita Bringmann ◽  
Lothar Kanz ◽  
Frank Grünebach ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Antigen Presenting Cells ◽  
Poly I:C ◽  
Blood Monocytes ◽  
Splice Form ◽  
Antigen Presenting

Abstract Dendritic cells play an inimitable role in the functioning immune system as they are the most potent antigen presenting cells being able to prime naive T-cells. Their characteristic properties that enable them to take up antigens and present them to leukocytes are due to an expression of specific genes and thus specific proteins that are unique to this subset of antigen-presenting cells. Using a substractive cDNA library based on suppression hybridization between DC cDNA and the reference monocyte cDNA, we identified in DC two differentially expressed genes p275 and p306. p275 codes for a membrane protein and represents a splice isoform of the transport protein NAT-1. The predicted structure of protein p306 is globular, suggesting that the protein is either intracellular or secreted. The expression of both genes was confirmed by RT-PCR using cDNA isolated from peripheral blood monocytes and DC, generated in vitro from monocytes or CD34+ progenitor cells. To further analyze the protein expression polyclonal antibodies were generated by immunization with synthetic peptides deduced from the identified sequences. Interestingly, inhibition of DC differentiation using IL-10 or STI571 (Imatinib) resulted in an impaired expression of both proteins. Utilizing specific primers for two recently described splice variants of p306 we identified a new splice form expressed in DC. While the gene of p306 contains eight exons, splice variant 1 consists of the exons 1,2,4,5,6, and 7 and splice variant 2 contains the exons 1,2,3,4,5,6, a shortened exon 7, and exon 8. The new identified splice form includes the exons 1–7. However, as the open reading frame starts in exon 4, the expressed protein is identical with the one corresponding to splice variant 1. Analyzing different DC populations in peripheral blood we show that p306 is expressed in plasmacytoid, but not myeloid DC. Interestingly, the activation of DC with Toll-like receptor ligands (TLRL) Pam3Cys (TLR2L), Poly I:C (TLR3L), LPS (TLR4L) and R848 (TLR7L) has no influence on the expression of p306. Although the functions of p275 and p306 in DC have yet to be determined, both genes play a role in DC differentiation and are found in different hematopoietic cell populations. Especially p306 might be an interesting marker of plasmacytoid DC as the predicted protein does not resemble any known protein structure.

scholarly journals A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

2021 ◽  
Author(s):  
Phillip C. Burke ◽  
Heungwon Park ◽  
Arvind Rasi Subramaniam
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Translational Control ◽  
Gc Content ◽  
Human Cells ◽  
Sequence Motifs ◽  
Coding Sequence ◽  
Ribosome Stalling ◽  
Nascent Peptide

AbstractStability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers acute mRNA instability. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro drive ribosome stalling and mRNA instability in vivo. The resulting nascent peptide code underlies ribosome stalling and mRNA-destabilizing effects of hundreds of endogenous peptide sequences in the human proteome. Our work reveals an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.

scholarly journals Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays

2009 ◽  
Vol 296 (6) ◽  
pp. F1396-F1404 ◽  
Author(s):  
Alfred N. Van Hoek ◽  
Richard Bouley ◽  
YingXian Lu ◽  
Claudia Silberstein ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
Collecting Duct ◽  
Dominant Role ◽  
Water Channel ◽  
Membrane Expression ◽  
Expression Levels ◽  
Stimulation Of

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK1 cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys8]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points ( day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 ∼ 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 → 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

Expression of fibronectin mRNA splice variants by rabbit lung in vivo and by alveolar type II cells in vitro

1996 ◽  
Vol 271 (6) ◽  
pp. L972-L980
Author(s):  
W. M. Maniscalco ◽  
R. H. Watkins ◽  
M. H. Campbell
Keyword(s):  
Gene Transcription ◽  
Splice Variant ◽  
Splice Variants ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Injured Lung

Fibronectin (FN) is a multidomain glycoprotein with putative functions in tissue development and repair. In repair of alveolar injury, FN may promote the transition of type II epithelial cells to type I epithelial cells. Alternative splicing of FN mRNA, including the EIIIA and EIIIB exons, results in protein isoforms that have cell, tissue, and developmental specificity. The present work found that FN mRNA with the EIIIA exon was in fetal, adult, and oxidant-injured lung. The EIIIB splice variant, however, was restricted to fetal lung and adult lung recovering from oxidant injury. Because alveolar type II cells in vitro express FN, we examined the splice variants in two conditions that induce FN [transforming growth factor-beta 1 (TGF-beta 1) treatment and time in culture]. TGF-beta 1 increased both EIIIA and EIIIB mRNA abundance by 10-fold. Increased EIIIA isoform immunostaining was also noted. Type II cells that spontaneously express FN at 72 h in vitro had increased EIIIA and EIIIB mRNA and increased immunostaining for EIIIA. Nuclear runoff showed induction of FN gene transcription at 72 h in vitro. Together, these data show differential FN splice variant expression in lung, with EIIIB mRNA restricted to fetal and recovering oxidant-injured lung. Furthermore, the transition of type II cells to a type I-like cell is accompanied by increased FN gene transcription and induction of both EIIIA and EIIIB mRNA.

scholarly journals Select amino acids in DGCR8 are essential for the UGU-pri-miRNA interaction and processing

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Thi Lieu Dang ◽  
Cong Truc Le ◽  
Minh Ngoc Le ◽  
Trung Duc Nguyen ◽  
Thuy Linh Nguyen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mirna Expression ◽  
Rna Binding ◽  
Human Cells ◽  
Current Understanding ◽  
Mirna Biogenesis ◽  
Cleavage Efficiency ◽  
Heme Domain

AbstractMicroprocessor, composed of DROSHA and DGCR8, processes primary microRNAs (pri-miRNAs) in miRNA biogenesis. Its cleavage efficiency and accuracy are enhanced because DGCR8 interacts with the apical UGU motif of pri-miRNAs. However, the mechanism and influence of DGCR8–UGU interaction on cellular miRNA expression are still elusive. In this study, we demonstrated that Rhed (i.e., the RNA-binding heme domain, amino acids 285–478) of DGCR8 interacts with UGU. In addition, we identified three amino acids 461–463 in Rhed, which are critical for the UGU interaction and essential for Microprocessor to accurately and efficiently process UGU-pri-miRNAs in vitro and UGU-miRNA expression in human cells. Furthermore, we found that within the DGCR8 dimer, the amino acids 461–463 from one monomer are capable of discriminating between UGU- and noUGU-pri-miRNAs. Our findings improve the current understanding of the substrate-recognizing mechanism of DGCR8 and implicate the roles of this recognition in differentiating miRNA expression in human cells.

scholarly journals The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

1996 ◽  
Vol 16 (8) ◽  
pp. 4052-4063 ◽  
Author(s):  
M L Schmitz ◽  
A Indorf ◽  
F P Limbourg ◽  
H Städtler ◽  
E B Traenckner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Splice Variant ◽  
Core Promoter ◽  
Adenovirus Type ◽  
Suppressive Effect ◽  
Oxygen Intermediates ◽  
Nf Kappab ◽  
Apoptotic Effects ◽  
Adenovirus Type 5

The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB.

scholarly journals Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin β subunits

2002 ◽  
Vol 156 (2) ◽  
pp. 361-376 ◽  
Author(s):  
Arjan van der Flier ◽  
Ingrid Kuikman ◽  
Duco Kramer ◽  
Dirk Geerts ◽  
Maaike Kreft ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
C2c12 Cells ◽  
Pcr Analysis ◽  
Filamin A ◽  
Filamentous Actin ◽  
Filamin B ◽  
High Affinity Binding Site ◽  
Mere Presence

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin β1A and β1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (ΔH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin β subunits. The mere presence of the high-affinity binding site for β1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(ΔH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

scholarly journals Characterization of Plasmodium berghei Pbg37 as Both a Pre- and Postfertilization Antigen with Transmission-Blocking Potential

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Fei Liu ◽  
Li Li ◽  
Wenqi Zheng ◽  
Yiwen He ◽  
Yaru Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasmodium Berghei ◽  
Protein Targeting ◽  
Immunofluorescence Assay ◽  
Specific Gene ◽  
Transmission Blocking ◽  
Content Type ◽  
Surface Localization

ABSTRACT Transmission-blocking vaccines (TBVs) interrupting malaria transmission are an integrated tool for malaria eradication. We characterized a sexual-stage-specific gene (PBANKA_060330) from Plasmodium berghei and studied its potential for use as a TBV. This gene, referred to as pbg37, encodes a protein of 37 kDa with a signal peptide and multiple transmembrane domains and is preferentially expressed in gametocytes. A recombinant Pbg37 (rPbg37) protein targeting the N-terminal 63 amino acids (amino acids 26 to 88) expressed in bacteria elicited strong antibody responses in mice. Western blotting demonstrated Pbg37 expression in gametocytes, zygotes, and, to a lesser extent, ookinetes and its predominant association with the membranes of gametocytes. Indirect immunofluorescence assay showed an abundant surface localization of Pbg37 on gametes and zygotes but reduced amounts on retorts and ookinetes. Knockout of pbg37 (Δpbg37) led to a considerable reduction in gametocytemia, which translated into a ~92.1% decrease in the oocyst number in mosquitoes. Deletion of pbg37 had a more substantial influence on the development and maturation of microgametocytes. As a result, the Δpbg37 lines exhibited a higher female/male gametocyte ratio, fewer mature male gametocytes, and defects in the exflagellation of mature microgametocytes. To test the transmission-blocking potential of Pbg37, an in vitro ookinete assay showed that the major inhibitory effects of anti-Pbg37 antiserum were on the exflagellation and fertilization processes. Direct feeding of mosquitoes on mice immunized with rPbg37 or a control protein showed that rPbg37-immunized and P. berghei-infected mice had a significant reduction (49.1%) in oocyst density compared to the controls. The conservation of this gene in Plasmodium warrants further investigations in human malaria parasites.

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