Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase

2019 ◽  
pp. 227-241 ◽  
Author(s):  
Michelle A. Sieburg ◽  
Elizabeth R. Cleverdon ◽  
James L. Hougland
Keyword(s):  
Biochemical Assays

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2021 ◽  
Vol 7 (5) ◽  
pp. eabc6479
Author(s):  
Sarah A. Mosure ◽  
Timothy S. Strutzenberg ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Laura A. Solt ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Binding Mode ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Heme Binding ◽  
Endogenous Ligand ◽  
Interaction Domain ◽  
Response Elements ◽  
Biochemical Assays ◽  
Chemical Cross Linking

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

scholarly journals Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 216
Author(s):  
Alanna C. Tseng ◽  
Vivek R. Nerurkar ◽  
Kabi R. Neupane ◽  
Helmut Kae ◽  
Pakieli H. Kaufusi
Keyword(s):  
West Nile Virus ◽  
Enzymatic Activity ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
West Nile ◽  
Biochemical Assays ◽  
In Trans ◽  
Viral Polyprotein ◽  
Ns3 Protease

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

scholarly journals PDGFRα: Expression and Function during Mitral Valve Morphogenesis

2021 ◽  
Vol 8 (3) ◽  
pp. 28
Author(s):  
Kelsey Moore ◽  
Diana Fulmer ◽  
Lilong Guo ◽  
Natalie Koren ◽  
Janiece Glover ◽  
...  
Keyword(s):  
Mitral Valve ◽  
Primary Cilia ◽  
Growth Factor Receptor ◽  
Akt Phosphorylation ◽  
Valve Development ◽  
Biochemical Assays ◽  
Factor Receptor Alpha ◽  
Mesenchymal Transformation ◽  
And Function

Mitral valve prolapse (MVP) is a common form of valve disease and can lead to serious secondary complications. The recent identification of MVP causal mutations in primary cilia-related genes has prompted the investigation of cilia-mediated mechanisms of disease inception. Here, we investigate the role of platelet-derived growth factor receptor-alpha (PDGFRα), a receptor known to be present on the primary cilium, during valve development using genetically modified mice, biochemical assays, and high-resolution microscopy. While PDGFRα is expressed throughout the ciliated valve interstitium early in development, its expression becomes restricted on the valve endocardium by birth and through adulthood. Conditional ablation of Pdgfra with Nfatc1-enhancer Cre led to significantly enlarged and hypercellular anterior leaflets with disrupted endothelial adhesions, activated ERK1/2, and a dysregulated extracellular matrix. In vitro culture experiments confirmed a role in suppressing ERK1/2 activation while promoting AKT phosphorylation. These data suggest that PDGFRα functions to suppress mesenchymal transformation and disease phenotypes by stabilizing the valve endocardium through an AKT/ERK pathway.

Role for Biochemical Assays and Kayser-Fleischer Rings in Diagnosis of Wilson’s Disease

2020 ◽  
Author(s):  
Yi Dong ◽  
Rou-Min Wang ◽  
Guo-Min Yang ◽  
Hao Yu ◽  
Wan-Qing Xu ◽  
...  
Keyword(s):  
Wilson’S Disease ◽  
Wilson's Disease ◽  
Biochemical Assays

scholarly journals Origins of human genetics. A personal perspective

2021 ◽  
Author(s):  
Eberhard Passarge
Keyword(s):  
Patient Care ◽  
Human Genetics ◽  
Cultured Cells ◽  
Genetic Research ◽  
Genetic Diagnosis ◽  
Population Level ◽  
Human Diseases ◽  
Personal Perspective ◽  
Biochemical Assays ◽  
American Society

AbstractGenetics evolved as a field of science after 1900 with new theories being derived from experiments obtained in fruit flies, bacteria, and viruses. This personal account suggests that the origins of human genetics can best be traced to the years 1949 to 1959. Several genetic scientific advances in genetics in 1949 yielded results directly relating to humans for the first time, except for a few earlier observations. In 1949 the first textbook of human genetics was published, the American Journal of Human Genetics was founded, and in the previous year the American Society of Human Genetics. In 1940 in Britain a textbook entitled Introduction to Medical Genetics served as a foundation for introducing genetic aspects into medicine. The introduction of new methods for analyzing chromosomes and new biochemical assays using cultured cells in 1959 and subsequent years revealed that many human diseases, including cancer, have genetic causes. It became possible to arrive at a precise cause-related genetic diagnosis. As a result the risk of occurrence or re-occurrence of a disease within a family could be assessed correctly. Genetic counseling as a new concept became a basis for improved patient care. Taken together the advances in medically orientated genetic research and patient care since 1949 have resulted in human genetics being both, a basic medical and a basic biological science. Prior to 1949 genetics was not generally viewed in a medical context. Although monogenic human diseases were recognized in 1902, their occurrence and distribution were considered mainly at the population level.

scholarly journals Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking

2007 ◽  
Vol 81 (21) ◽  
pp. 12019-12028 ◽  
Author(s):  
Hilde M. van der Schaar ◽  
Michael J. Rust ◽  
Barry-Lee Waarts ◽  
Heidi van der Ende-Metselaar ◽  
Richard J. Kuhn ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
High Ratio ◽  
Cell Entry ◽  
Viral Membrane ◽  
Virus Particles ◽  
Biochemical Assays ◽  
Particle Ratio ◽  
Infectious Unit

ABSTRACT In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.

scholarly journals P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation

2021 ◽  
Vol 22 (12) ◽  
pp. 6542
Author(s):  
Kate Dunning ◽  
Adeline Martz ◽  
Francisco Andrés Peralta ◽  
Federico Cevoli ◽  
Eric Boué-Grabot ◽  
...  
Keyword(s):  
Single Channel ◽  
Cell Membrane Permeability ◽  
Large Molecule ◽  
Cholesterol Depletion ◽  
Open Probability ◽  
P2x7 Receptors ◽  
Biochemical Assays ◽  
Functional Complex ◽  
Macropore Formation ◽  
Current Facilitation

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

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