scholarly journals Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis

2019 ◽  
Vol 7 (4) ◽  
Author(s):  
Bradley L. Hoare ◽  
Martina Kocan ◽  
Shoni Bruell ◽  
Daniel J. Scott ◽  
Ross A. D. Bathgate
Keyword(s):  
Cell Surface ◽  
Label Cell ◽  
The Novel ◽  
Proximity Analysis

scholarly journals ACE2 Nascence, trafficking, and SARS-CoV-2 pathogenesis: the saga continues

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Sally Badawi ◽  
Bassam R. Ali
Keyword(s):  
Cell Surface ◽  
Angiotensin Converting Enzyme ◽  
The Novel ◽  
Converting Enzyme ◽  
Post Translational Modifications ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Novel Coronavirus ◽  
Effective Medication ◽  
Potential Use

AbstractWith the emergence of the novel coronavirus SARS-CoV-2 since December 2019, more than 65 million cases have been reported worldwide. This virus has shown high infectivity and severe symptoms in some cases, leading to over 1.5 million deaths globally. Despite the collaborative and concerted research efforts that have been made, no effective medication for COVID-19 (coronavirus disease-2019) is currently available. SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as an initial mediator for viral attachment and host cell invasion. ACE2 is widely distributed in the human tissues including the cell surface of lung cells which represent the primary site of the infection. Inhibiting or reducing cell surface availability of ACE2 represents a promising therapy for tackling COVID-19. In this context, most ACE2–based therapeutic strategies have aimed to tackle the virus through the use of angiotensin-converting enzyme (ACE) inhibitors or neutralizing the virus by exogenous administration of ACE2, which does not directly aim to reduce its membrane availability. However, through this review, we present a different perspective focusing on the subcellular localization and trafficking of ACE2. Membrane targeting of ACE2, and shedding and cellular trafficking pathways including the internalization are not well elucidated in literature. Therefore, we hereby present an overview of the fate of newly synthesized ACE2, its post translational modifications, and what is known of its trafficking pathways. In addition, we highlight the possibility that some of the identified ACE2 missense variants might affect its trafficking efficiency and localization and hence may explain some of the observed variable severity of SARS-CoV-2 infections. Moreover, an extensive understanding of these processes is necessarily required to evaluate the potential use of ACE2 as a credible therapeutic target.

scholarly journals ACE2 Nascence, Trafficking and SARS-CoV-2 Pathogenesis: The Saga Continues

2020 ◽  
Author(s):  
Sally Badawi ◽  
Bassam Ali
Keyword(s):  
Cell Surface ◽  
Angiotensin Converting Enzyme ◽  
Virus Disease ◽  
The Novel ◽  
Converting Enzyme ◽  
Post Translational Modifications ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Corona Virus ◽  
Potential Use

With the emergence of the novel corona virus SARS-CoV-2 since December 2019, more than 43 million cases have been reported worldwide. This virus has shown high infectivity and severe symptoms in some cases leading to over 1 million deaths globally. Despite the collaborative and concerted research efforts that has been made, no effective treatment for COVID-19 (corona virus disease-2019) is currently available. SARS-CoV-2 uses the angiotensin converting enzyme 2 (ACE2) as an initial mediator for viral attachment and host cell invasion. ACE2 is widely distributed in human tissues including the cell surface of lung cells which represent the primary site of the infection. Inhibiting or reducing cell surface availability of ACE2 represents a promising therapy for tackling COVID-19. In this context, most ACE2–based therapeutic strategies have aimed to achieve this through the use of angiotensin converting enzyme (ACE) inhibitors or neutralizing the virus by exogenous administration of ACE2. However, through this review, we present another perspective focusing on the subcellular localization and trafficking of ACE2. Membrane targeting of ACE2, shedding and its cellular trafficking pathways including internalization are not well elucidated. Therefore, hereby we present an overview on the fate of newly synthesized ACE2, its post translational modifications, what is known of its trafficking pathways. In addition, we highlight the possibility that some of the identified ACE2 missense variants might affect its trafficking efficiency and localization and hence may explain some of the observed variable severity of SARS-CoV-2 infections. Extensive understanding of these processes is necessary to evaluate the potential use of ACE2 as a credible therapeutic target.

scholarly journals The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation, Cell Surface Expression, and Secretion of the Amyloid Precursor Protein

2010 ◽  
Vol 285 (27) ◽  
pp. 20664-20674 ◽  
Author(s):  
Sylvia Ullrich ◽  
Anna Münch ◽  
Stephanie Neumann ◽  
Elisabeth Kremmer ◽  
Jörg Tatzelt ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Surface Expression ◽  
Precursor Protein ◽  
Cell Surface Expression ◽  
The Novel

scholarly journals Immunophenotypic Analysis of B Lymphocytes in Patients with Common Variable Immunodeficiency: Identification of CD23 as a Useful Marker in the Definition of the Disease

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Giuseppe Patuzzo ◽  
Filippo Mazzi ◽  
Antonio Vella ◽  
Riccardo Ortolani ◽  
Alessandro Barbieri ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Lymphocytes ◽  
Clinical Features ◽  
Common Variable Immunodeficiency ◽  
The Novel ◽  
Cell Surface Molecules ◽  
Surface Molecules ◽  
Definition Of ◽  
Common Variable

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by the failure of B lymphocytes differentiation leading to deficient immunoglobulins secretion. The identified genetic defects account only for a minority of cases. The importance of B cells immunophenotyping in the classification of CVID is known. This procedure can identify alterations on the cell surface molecules expression that could explain some immunological disorders characteristic of CVID. Moreover, some immunophenotypical aspects can correlate with clinical features of the disease. We used this procedure to analyze a cohort of 23 patients affected by CVID, in order to identify the novel alterations of B cells and to find the possible correlations with clinical features. Circulating B cells were studied by flow cytometry incubating whole blood with specific antibodies for B cell surface molecules including CD27, IgM, IgD, CD21, and CD23. We compared the population of “switched memory” IgD− CD27+ B lymphocytes with the population of “switched memory” IgM− IgD− CD23− CD27+ B cells. These last B cells were reduced in patients compared to healthy controls; moreover, IgM− IgD− CD23− CD27+ B cells were lower than IgD− CD27+ B cells in patients with CVID. The reduction of this subset of B lymphocytes correlates more tightly than IgD− CD27+ B cells with lymphadenopathy and airways infections. In conclusion, our findings may help in better identifying patients with CVID.

scholarly journals Discovery of Novel IL3RA (CD123) Isoforms By Long Read Transcriptomics, Heterogeneous Expression Among AML Patient Cohorts and the Implications for Anti-CD123 Therapeutics

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4658-4658 ◽  
Author(s):  
Jason G Underwood ◽  
Jenny L. Smith ◽  
Lindsey F. Call ◽  
Elizabeth Tseng ◽  
Tiffany A. Hylkema ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Peptide Sequence ◽  
The Novel ◽  
Short Read ◽  
Rnaseq Data ◽  
Wide Range ◽  
Long Read ◽  
Pediatric Aml ◽  
Novel Isoforms

CD123 is a cell surface protein expressed on hematopoietic progenitors and the surface of most AML blasts, making it a valuable therapeutic target for clinical intervention. As such, antibody-drug conjugates or CAR T cells against this antigen have been developed including tagraxofusp-erzs, recently approved for blastic plasmacytoid dendritic cell neoplasm (BPDCN). CD123 is the alpha subunit of the interleukin 3 receptor and is encoded by the pseudoautosomal IL3RA gene. Recent work demonstrated that different monoclonal antibodies directed against CD123 show sizable discrepancies when used to quantify this antigen on AML patient samples. (Cruz et al. 2018) Given these results and the variability in patient response to anti-CD123 therapeutics, we hypothesized that heterogeneity in IL3RA mRNA isoform expression may induce epitope variation on the cell surface, modulating antibody and therapeutic response. To better understand the heterogeneity, we analyzed long and short read transcriptomics data from normal bone marrow along with pediatric AML samples known to harbor translocations. The combination of these two types of RNA expression data afford both a look at full length isoforms produced in patients and the relative expression levels of each. To define the isoforms expressed in pediatric AML, we augmented short read RNAseq with long read transcriptomics on the PacBio platform. Following up on short RNAseq data generated from 4 clinical study cohorts of pediatric AML samples (N = 1,394) collected and normal bone marrow controls (NBM, N = 68), we chose diagnostic AML samples (N=10) and one NBM with high RNA integrity (RIN >9) for polyA transcript profiling using Pacific Biosciences (PacBio) long read RNA sequencing. This method gives full isoform sequences that can be reliably translated into open reading frames. It also adds new utility to our wealth of short read RNA-seq as the long read data can be used in a reference fashion to quantify and compare isoforms across cohorts. After profiling and classifying the novel isoforms, we honed in on transcripts from the IL3RA locus since these encode the CD123 antigen targeted by immunotherapy approaches. PacBio long read RNA sequencing detected 8 unique full-length transcript isoforms that mapped to the IL3RA gene: 4 known and 4 novel IL3RA transcripts. Three abundant known isoforms aligned to the canonical annotated IL3RA (Isoform 1, Figure 1A), an isoform missing exons 3 and 4 (Isoform 2) or a third isoform (Isoform 3, not shown) which does not encode a transmembrane domain. We focused on 3 novel isoforms (Figure 1, Isoforms A-C) encompassing a variety of splicing changes, but all of which are predicted to harbor a transmembrane domain and dramatically alter the extracellular peptide sequence in comparison to annotated isoforms. (Figure 1, domains predicted and colored in the legend) The novel isoforms were found independently in multiple patients, but as additional validation we PCR amplified cDNA from patient samples using an inclusive primer set directed to constitutive exons that flank the alternative splicing events and thus designed to capture multiple isoforms. (Figure 2A, arrows) Products were separated by gel electrophoresis with amplicons cloned, Sanger sequenced and analyzed through alignment with human reference sequences. The non-specific isoform amplification detects multiple isoforms indicating heterogeneity in splice site choice between patients. Fragment analysis from patient 2 (Figure 2B) confirms the presence of isoform variation with peaks corresponding with the expected products from isoforms 1, 2, A, B, and C. In an effort to further validate and quantify novel isoforms of IL3RA, we employed kallisto which utilizes short read RNAseq data from the entire cohort to get a count estimate for each isoform in pediatric AML patient samples and normal controls. These data (Figure 3) indicate that while the annotated isoform 1 is the most abundant, a wide range of novel isoform expression is detected in both normal and pAML samples. In conclusion, changes in protein length and peptide sequence may affect the efficacy of therapeutic anti-CD123 approaches since some patients express alternative isoforms with a wide range of abundance. We anticipate that the computational and experimental pipeline used to discover and characterize these isoforms will be of high value in the study of many cell surface antigens with therapeutic potential. Disclosures Underwood: Pacific Biosciences: Employment, Equity Ownership. Tseng:Pacific Biosciences: Employment, Equity Ownership. Farrar:Novartis: Research Funding.

scholarly journals Mosquito defensins enhance Japanese encephalitis virus infection by facilitating virus adsorption and entry within mosquito

2020 ◽  
Author(s):  
Ke Liu ◽  
Changguang Xiao ◽  
Shumin Xi ◽  
Muddassar hameed ◽  
Abdul Wahaab ◽  
...  
Keyword(s):  
Cell Surface ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Target Cells ◽  
The Novel ◽  
Antiviral Protein ◽  
Japanese Encephalitis Virus Infection ◽  
Virus Adsorption ◽  
Lipoprotein Receptor Related Protein

AbstractJapanese encephalitis virus (JEV) is a viral zoonosis which can cause viral encephalitis, death and disability. Culex is the main vector of JEV, but little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via both direct and indirect pathways. Mosquito defensin bound the ED III domain of viral E protein and directly mediated efficient virus adsorption on the target cell surface, Lipoprotein receptor-related protein 2 expressed on the cell surface is the receptor affecting defensin dependent adsorption. Mosquito defensin also indirectly down-regulated the expression of an antiviral protein, HSC70B. As a result, mosquitos defensin enhances JEV infection in salivary gland while increasing the possibility of viral transmission by mosquito. These findings demonstrate that the novel effects of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.

Revising the Genomic Structure of the Human GM-CSF Receptor Alpha (GMRα) Subunit: Characterization of a Novel Membrane-Spanning GMRα Isoform Which Contains a Previously Undescribed Exon.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2167-2167
Author(s):  
Jennifer L. Pelley ◽  
Chris D. Nicholls ◽  
Tara L. Beattie ◽  
Christopher B. Brown
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Cell Types ◽  
Extracellular Domain ◽  
Hematopoietic Cell ◽  
Conditioned Media ◽  
Full Length ◽  
Human Neutrophils ◽  
The Novel ◽  
Gm Csf

Abstract The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key player in the processes of hematopoiesis and inflammation. The functional effects of GM-CSF are mediated by binding of the cytokine to the cell surface GM-CSF receptor, which is comprised of a ligand-binding alpha subunit (GMRα) and a signal-transducing beta subunit (GMRβ). In addition, there are at least 2 soluble GMRα isoforms (sGMRα)-one generated by alternative splicing, and a second produced by proteolytic release of the GMRα extracellular domain from the cell surface. While examining expression of the spliced soluble and transmembrane (tmGMRα) isoforms of GMRα in neutrophils by RT-PCR, we detected a third, higher molecular weight, GMRα transcript. This was surprising since the tmGMRα transcript is comprised of all 13 exons of the published GMRα gene structure (Nakagawa et al., 1994). We therefore hypothesized that we had discovered a previously undescribed exon of the GMRα gene. The novel GMRα (nGMRα) transcript was subsequently cloned from human neutrophils. Sequencing of nGMRα indicated inclusion of a 102 nucleotide sequence between exons 10 and 11 of the GMRα gene. Interestingly, the novel exon and surrounding intronic sequence appear to represent an Alu-repeat element, indicating that this exon is likely primate-specific. Segments of these repetitive DNA elements become inserted into mature mRNAs by way of splicing in a process termed “exonization”. In fact, it now seems that more than 5% of the alternatively spliced exons in the human genome are Alu-derived. At the protein level, this novel transcript is predicted to contain all 400 amino acids of the tmGMRα protein as well as an additional 34 amino acids within the membrane-proximal region of the extracellular domain. We have expressed the novel GMRα clone, as well as a soluble version of novel GMRα (sol-nGMRα), in the murine factor-dependent cell line Ba/F3. The sol-nGMRα protein is secreted from these cells into conditioned media, as expected. We have purified sol-nGMRα protein and used it to determine the affinity of nGMRα for GM-CSF. By flow cytometry, we were unable to detect full-length nGMRα on the surface of Ba/F3 cells, but we were able to detect nGMRα in conditioned media from nGMRα-expressing Ba/F3 cells. In addition, while tmGMRα-expressing Ba/F3 cells are able to proliferate in response to human GM-CSF in the absence of murine growth factors, the nGMRα-expressing Ba/F3 cells do not respond to GM-CSF. Our results indicate that the full-length nGMRα protein is proteolytically cleaved from the cell surface or alternatively, that its subcellular localization is otherwise disrupted. As the nGMRα transcript was first detected in neutrophils isolated from a human donor, we sought to determine whether nGMRα would be expressed by other donors and in other hematopoietic cell types. Our results indicate that all donors tested express nGMRα, and that nGMRα is present in all hematopoietic cell types expressing the tmGMRα transcript, although tmGMRα nonetheless represents the predominant transcript. It is becoming increasingly clear that GM-CSF signaling is more complex than was previously thought. In order to understand the actions of GM-CSF in the clinical setting, it is critical that we first define all components of the GM-CSF signaling axis.

Specific Interaction between an Oligosaccharide on the Tumor Cell Surface and the Novel Antitumor Agents, Sulfoquinovosylacylglycerols

2001 ◽  
Vol 288 (4) ◽  
pp. 893-900 ◽  
Author(s):  
Keisuke Ohta ◽  
Yoshiyuki Mizushina ◽  
Takayuki Yamazaki ◽  
Shinya Hanashima ◽  
Fumio Sugawara ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tumor Cell ◽  
Antitumor Agents ◽  
Specific Interaction ◽  
The Novel
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