scholarly journals Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

2020 ◽  
Author(s):  
Dorothea Höpfner ◽  
Joel Fauser ◽  
Marietta S. Kaspers ◽  
Christian Pett ◽  
Christian Hedberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Recent Progress ◽  
Regulatory Mechanism ◽  
Target Identification ◽  
Adenosine Monophosphate ◽  
Post Translational Modification ◽  
Highly Sensitive ◽  
Amino Acid Side Chains

AbstractAMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

scholarly journals Evolution of molecular determinants for SUMO-activating enzyme subcellular localization in plants

2020 ◽  
Author(s):  
Abraham Más ◽  
Laura Castaño-Miquel ◽  
Lorenzo Carretero-Paulet ◽  
Núria Colomé ◽  
Francesc Canals ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Regulatory Mechanism ◽  
Large Subunit ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
Sumo Conjugation ◽  
Phylogenetic Studies ◽  
Molecular Determinants ◽  
Evolutionary Role

AbstractPost-translational modification by Small Ubiquitin-related Modifier (SUMO) is an essential regulatory mechanism in eukaryotes. In the cell, SUMO conjugates are highly enriched in the nucleus and, consistently, SUMOylation machinery components are mainly nuclear. Nonetheless, cytosolic SUMO targets also exist and the mechanisms that facilitate SUMO conjugation in the cytosol are unknown. Here, we show that the nuclear localization of the Arabidopsis SUMO activating enzyme large subunit SAE2 is dependent on two nuclear localization signals, the canonical NLS1 and the non-canonical NLS2 identified and validated here. NLS2 is proteolytic processed from SAE2 during seed development, facilitating SAE2 enrichment in the cytosol. Results obtained using transgenic plants expressing different SAE2 proteoforms suggest that SAE2 cytosolic enrichment could constitute a rapid signal for growth arrest. Phylogenetic studies indicated that the Arabidopsis NLS1-NLS2 structural organization is conserved only in seed plants, providing a potential evolutionary role of cytosolic SUMOylation in seed appearance.

Role of charged amino acid side chains in the stability and lipid binding ofManduca sexta apolipophorin III

1995 ◽  
Vol 30 (2-3) ◽  
pp. 211-223 ◽  
Author(s):  
Minal Upadhyaya ◽  
Kim Oikawa ◽  
Cyril M. Kay ◽  
Douglas G. Scraba ◽  
Roger Bradley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lipid Binding ◽  
Side Chains ◽  
Apolipophorin Iii ◽  
Amino Acid Side Chains ◽  
The Stability

scholarly journals In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Angelica Aguilera-Gomez ◽  
Marinke M van Oorschot ◽  
Tineke Veenendaal ◽  
Catherine Rabouille
Keyword(s):  
Amino Acid ◽  
Metabolic Stress ◽  
Amino Acid Starvation ◽  
Critical Event ◽  
Post Translational Modification ◽  
Adp Ribosylation ◽  
Body Component ◽  
Necessary And Sufficient

PARP catalysed ADP-ribosylation is a post-translational modification involved in several physiological and pathological processes, including cellular stress. In order to visualise both Poly-, and Mono-, ADP-ribosylation in vivo, we engineered specific fluorescent probes. Using them, we show that amino-acid starvation triggers an unprecedented display of mono-ADP-ribosylation that governs the formation of Sec body, a recently identified stress assembly that forms in Drosophila cells. We show that dPARP16 catalytic activity is necessary and sufficient for both amino-acid starvation induced mono-ADP-ribosylation and subsequent Sec body formation and cell survival. Importantly, dPARP16 catalyses the modification of Sec16, a key Sec body component, and we show that it is a critical event for the formation of this stress assembly. Taken together our findings establish a novel example for the role of mono-ADP-ribosylation in the formation of stress assemblies, and link this modification to a metabolic stress.

The role of amino acid side chains in stabilizing dipeptides: the laser ablation Fourier transform microwave spectrum of Ac-Val-NH2

2017 ◽  
Vol 19 (36) ◽  
pp. 24985-24990 ◽  
Author(s):  
I. León ◽  
E. R. Alonso ◽  
S. Mata ◽  
C. Cabezas ◽  
M. A. Rodríguez ◽  
...  
Keyword(s):  
Fourier Transform ◽  
Amino Acid ◽  
Laser Ablation ◽  
Molecular Beam ◽  
Microwave Spectrum ◽  
Steric Effects ◽  
Side Chains ◽  
Isopropyl Group ◽  
Amino Acid Side Chains

The steric effects imposed by the isopropyl group of valine in the conformational stabilization of the capped dipeptide N-acetyl-l-valinamide (Ac-Val-NH2) have been studied by laser ablation molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopy.

scholarly journals Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels

1990 ◽  
Vol 58 (6) ◽  
pp. 1367-1375 ◽  
Author(s):  
M.T. Tosteson ◽  
O. Alvarez ◽  
W. Hubbell ◽  
R.M. Bieganski ◽  
C. Attenbach ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Primary Structure ◽  
Voltage Gating ◽  
Side Chains ◽  
Amino Acid Side Chains

scholarly journals Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening

2012 ◽  
Vol 17 (7) ◽  
pp. 993-998 ◽  
Author(s):  
Kris F. Sachsenmeier ◽  
Carl Hay ◽  
Erin Brand ◽  
Lori Clarke ◽  
Kim Rosenthal ◽  
...  
Keyword(s):  
Malachite Green ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Performance ◽  
Adenosine Monophosphate ◽  
Screening Assay ◽  
Highly Sensitive ◽  
Development And Validation ◽  
Multiple Samples

5′-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.

The Functional Role of Positively Charged Amino Acid Side Chains in α-Bungarotoxin Revealed by Site-Directed Mutagenesis of a His-Tagged Recombinant α-Bungarotoxin†

Biochemistry ◽  
1999 ◽  
Vol 38 (24) ◽  
pp. 7847-7855 ◽  
Author(s):  
Julie A. Rosenthal ◽  
Mark M. Levandoski ◽  
Belle Chang ◽  
Jerald F. Potts ◽  
Qing-Luo Shi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Amino Acid Side Chains ◽  
Positively Charged

scholarly journals Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions

2017 ◽  
Author(s):  
Merve S. Zeden ◽  
Christopher F. Schuster ◽  
Lisa Bowman ◽  
Qiyun Zhong ◽  
Huw D. Williams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Adenosine Monophosphate ◽  
Defined Medium ◽  
Growth Defect ◽  
Uptake System ◽  
Osmotic Regulation ◽  
Bacterial Cells ◽  
Rich Medium ◽  
Anaerobic Conditions

ABSTRACTCyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such asStaphylococcus aureus. However, the exact role of this molecule has not been identified.dacA, theS. aureusgene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that anS. aureus dacAmutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality ofdacAwere identified inalsTandopuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system inS. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained inhepS, hemB, ctaAandqoxB, coding for proteins required for respiration. Furthermore, we show thatdacAis dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration inS. aureus.

The role of protein acetylation in regulating mitochondrial fusion and fission

2021 ◽  
Author(s):  
Golam M. Uddin ◽  
Rafa Abbas ◽  
Timothy E. Shutt
Keyword(s):  
Mitochondrial Function ◽  
Regulatory Mechanism ◽  
Mitochondrial Fusion ◽  
Dynamic Processes ◽  
Protein Acetylation ◽  
Post Translational Modification ◽  
Acetylation Status

The dynamic processes of mitochondrial fusion and fission determine the shape of mitochondria, which can range from individual fragments to a hyperfused network, and influence mitochondrial function. Changes in mitochondrial shape can occur rapidly, allowing mitochondria to adapt to specific cues and changing cellular demands. Here, we will review what is known about how key proteins required for mitochondrial fusion and fission are regulated by their acetylation status, with acetylation promoting fission and deacetylation enhancing fusion. In particular, we will examine the roles of NAD+ dependant sirtuin deacetylases, which mediate mitochondrial acetylation, and how this post-translational modification provides an exquisite regulatory mechanism to co-ordinate mitochondrial function with metabolic demands of the cell.

scholarly journals LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

2020 ◽  
Author(s):  
Jixin Shou ◽  
Haidong Gao ◽  
Sen Cheng ◽  
Bingbing Wang ◽  
Haibo Guan
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Regulatory Mechanism ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Brdu Assay ◽  
Rip Assay

Abstract Background: LncRNA HOXA-AS2 has been found in the literature to deteriorate glioblastoma. However, its regulatory mechanism is yet to be fully investigated. Our study focused chiefly on the interaction and role of the HOXA-AS2/miR-885-5p/RBBP4 axis in the development of glioblastoma. Methods: qRT-PCR analysis was performed to detect the expression of lncRNA, miRNA and mRNA in glioblastoma tissues and cells. Dual-luciferase assay, RIP assay and RNA pull-down assay were later carried out to reveal the interactions among HOXA-AS2, miR-885-5p and RBBP4. After that, CCK-8 assay, BrdU assay, nude mice xenografting assay, western blot assay, and flow cytometry were carried out to analyze the effect of the HOXA-AS2/miR-885-5p/RBBP4 axis on glioblastoma samples. Results: HOXA-AS2 and RBBP4 were found to be overexpressed in glioblastoma. Experimental results showed that HOXA-AS2 and RBBP4 contributed to the tumorigenesis of glioblastoma cells. However, miR-885-5p was observed to be downregulated in glioblastoma. Findings also indicated that HOXA-AS2 could negatively regulate miR-885-5p, thereby enhancing RBBP4 expression. Conclusion: Overall, HOXA-AS2 promoted the tumorigenesis of glioblastoma by targeting and regulating miR-885-5p to induce the expression of RBBP4.

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