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Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with (44)Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after (44)Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of (44)Sc-CHX-A″-DTPA-Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using (44)Sc-CHX-A″-DTPA-Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.
Post-translational modifications play important roles in controlling protein function and can lead to altered protein stability. Protein stability can be determined after treatment with the protein synthesis inhibitor Cycloheximide. Cycloheximide is a translational inhibitor that inhibits protein synthesis via cytoplasmic ribosomes. Here we describe how to measure the stability of MYC2 in the context of regulation by FERONIA receptor kinase. First, we describe how to measure MYC2 stability in wild-type and feronia mutant; then we describe similar assays in transgenic plants expressing MYC2-FLAG and MYC2A12-FLAG (12 FERONIA phosphorylation sites are mutated to Alanine and the mutant protein is stabilized). MYC2 can be induced by mechanical touch, which can be a confounding factor in protein level measurement. In this protocol, we take that into consideration and try to achieve more accurate measurement.
Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies.
ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other's expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins.
Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay. Graphical overview.
Short half-life is one of the key challenges in the field of therapeutic peptides. Various studies have reported enhancement in the stability of peptides using methods like chemical modifications, D-amino acid substitution, cyclization, replacement of labile aminos acids, etc. In order to study this scattered data, there is a pressing need for a repository dedicated to the half-life of peptides. To fill this lacuna, we have developed PEPlife (http://crdd.osdd.net/raghava/peplife), a manually curated resource of experimentally determined half-life of peptides. PEPlife contains 2229 entries covering 1193 unique peptides. Each entry provides detailed information of the peptide, like its name, sequence, half-life, modifications, the experimental assay for determining half-life, biological nature and activity of the peptide. We also maintain SMILES and structures of peptides. We have incorporated web-based modules to offer user-friendly data searching and browsing in the database. PEPlife integrates numerous tools to perform various types of analysis such as BLAST, Smith-Waterman algorithm, GGSEARCH, Jalview and MUSTANG. PEPlife would augment the understanding of different factors that affect the half-life of peptides like modifications, sequence, length, route of delivery of the peptide, etc. We anticipate that PEPlife will be useful for the researchers working in the area of peptide-based therapeutics.
Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Traditionally, such approaches have largely relied on random mutagenesis and display formats, which fail to address related critical attributes of the antibody, such as effector functions or biophysical stability. We have developed a structure- and network-based framework to interrogate the engagement of IgG with multiple Fc receptors (FcRn, C1q, TRIM21, FcγRI, FcγRIIa/b, FcγRIIIa) simultaneously. Using this framework, we identified features that govern Fc-FcRn interactions and identified multiple distinct pathways for enhancing FcRn binding in a pH-specific manner. Network analysis provided a novel lens to study the allosteric impact of half-life-enhancing Fc mutations on FcγR engagement, which occurs distal to the FcRn binding site. Applying these principles, we engineered a panel of unique Fc variants that enhance FcRn binding while maintaining robust biophysical properties and wild type-like binding to activating receptors. An antibody harboring representative Fc designs demonstrates a half-life improvement of > 9 fold in transgenic mice and > 3.5 fold in cynomolgus monkeys, and maintains robust effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
The proteasome controls the concentrations of most proteins in eukaryotic cells. It recognizes its protein substrates through ubiquitin tags and initiates degradation at disordered regions within the substrate. Here we show that the proteasome has pronounced preferences for the amino acid sequence of the regions at which it initiates degradation. Specifically, proteins in which the initiation regions have biased amino acid compositions show longer half-lives in yeast than proteins with unbiased sequences in the regions. The relationship is also observed on a genomic scale in mouse cells. These preferences affect the degradation rates of proteins in vitro, can explain the unexpected stability of natural proteins in yeast and may affect the accumulation of toxic proteins in disease. We propose that the proteasome's sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.
African swine fever virus is transmissible through animal consumption of contaminated feed. To determine virus survival during transoceanic shipping, we calculated the half-life of the virus in 9 feed ingredients exposed to 30-day shipment conditions. Half-lives ranged from 9.6 to 14.2 days, indicating that the feed matrix environment promotes virus stability.
Protein degradation is critical for maintaining cellular homeostasis. The 20S proteasome is selective for unfolded, extended polypeptide chains without ubiquitin tags. Sequestration of such segments by protein partners, however, may provide a regulatory mechanism. Here we used the AP-1 complex to study how c-Fos turnover is controlled by interactions with c-Jun. We show that heterodimerization with c-Jun increases c-Fos half-life. Mutations affecting specific contact sites (L165V, L172V) or charge separation (E175D, E189D, K190R) with c-Jun both modulate c-Fos turnover, proportionally to their impact on binding affinity. The fuzzy tail beyond the structured b-HLH/ZIP domain (~165 residues) also contributes to the stabilization of the AP-1 complex, removal of which decreases c-Fos half-life. Thus, protein turnover by 20S proteasome is fine-tuned by both specific and fuzzy interactions, consistently with the previously proposed "nanny" model.
mRNA decay is an important process in post-transcriptional regulation; in addition, it plays a crucial role in plant development and response to stress. The development of new tools to quantify mRNA decay intermediates is thus important to better characterize the dynamic of mRNA decay in various conditions. Here, we applied droplet digital PCR (ddPCR), a recent and precise PCR technology, to determine mRNA half-life in Arabidopsis seedlings. We demonstrated that ddPCR can correctly assess mRNA half-life from a wide variety of transcripts in a reproducible manner. We also demonstrated that thanks to multiplexing mRNA, the half-life of multiple transcripts can be followed in the same reaction. As ddPCR allows precise quantification, we proposed that this approach is highly suitable when a low amount of RNA is available; for the detection of many targets or for the analysis of lowly expressed transcripts.
System-wide quantitative characterization of human neonatal Fc receptor (FcRn) properties is critical for understanding and predicting human PK (pharmacokinetics) as well as the distribution of mAbs and Fc-fusion proteins using PBPK (physiologically-based pharmacokinetic) modeling. To this end, tissue-specific FcRn expression and half-life are important model inputs. Herein, human FcRn tissue expression was measured by peptide immunoaffinity chromatography coupled with high-resolution mass spectrometry. FcRn concentrations across 14 human tissues ranged from low to 230 pmol per gram of tissue. Furthermore, the FcRn half-life was determined to be 11.1 h from a human stable isotope labelled leucine pulse labeling experiment. The spatial and temporal quantitative human FcRn data now promise to enable a refined PBPK model with improved accuracy of human PK predictions for Fc-containing biotherapeutics.
Integrin αIIbβ₃ plays a pivotal role in platelet aggregation. Three αIIbβ₃ antagonists have been approved by the Food and Drug Administration (FDA) for the treatment of cardiovascular diseases. Unfortunately, all of these three drugs can cause the side effect of severe bleeding. Therefore, developing a new αIIbβ₃ antagonist with low bleeding was needed. In the present study, we screened compounds by using a fibrinogen/integrin αIIbβ₃ enzyme-linked immunosorbent assay (ELISA), and a novel αIIbβ₃ antagonist ANTP266 was attained. The antithrombotic effects of ANTP266 were estimated by using two animal models, the bleeding risk was estimated by using a mice tail cutting assay, and the plasma half-life time was tested by LC-MS/MS. The results showed that ANTP266 potently decreased thrombosis formation, while not prolonging bleeding time at its effective dosage. The bleeding of ANTP266 reduced rapidly as time went on from 5 to 60 min, but tirofiban produced high bleeding continuously. The plasma half-life of ANTP266 in rats was 10.8 min. Taken together, ANTP266 is an effective antithrombotic agent with a low bleeding risk. The shorter bleeding time benefits from its short plasma half-life. ANTP266 could be a candidate for developing the αIIbβ₃ antagonist of rapid elimination for a patient undergoing percutaneous coronary intervention.
Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.
The adipokine hormone, leptin, is a major component of body weight homeostasis. Numerous studies have been performed administering recombinant mouse leptin as an experimental reagent; however, the half-life of circulating leptin following exogenous administration of recombinant mouse leptin has not been carefully evaluated.
Glucagon-like peptide-1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) has been used widely for half-life extension of therapeutic peptides and proteins. In the present study, novel GLP-1 receptor agonists were designed by genetic fusion of GLP-1 to three kinds of ABDs with different affinities for HSA: GA3, ABD035 and ABDCon. The bioactivities and half-lives of ABD-fusion GLP-1 proteins with different types and lengths of linkers were investigated in vitro and in vivo. The results demonstrated that ABD-fusion GLP-1 proteins could bind to HSA with high affinity. The blood glucose-lowering effect of GLP-1 was significantly improved and sustained by fusion to ABD. Meanwhile, the fusion proteins significantly inhibited food intake, which was beneficial for T2DM and obesity treatment. The half-life of GLP-1 was substantially extended by virtue of ABD. The in vivo results also showed that a longer linker inserted between GLP-1 and ABD resulted in a higher blood glucose-lowering effect. The fusion proteins generated by fusion of GLP-1 to GA3, ABD035 and ABDCon exhibited similar bioactivities and pharmacokinetics in vivo. These findings demonstrate that ABD-fusion GLP-1 proteins retain the bioactivities of natural GLP-1 and can be further developed for T2DM treatment and weight loss. It also indicates that the ABD-fusion strategy can be generally applicable to any peptide or protein, to improve pharmacodynamic and pharmacokinetic properties.
RNA levels in a cell are regulated by the relative rates of RNA synthesis and decay. We recently developed a new approach for measuring both RNA synthesis and decay in a single experimental setting by biosynthetic labeling of newly transcribed RNA. Here, we show that this provides measurements of RNA half-lives from microarray data with a so far unreached accuracy. Based on such measurements of RNA half-lives for human B-cells and mouse fibroblasts, we identified conserved regulatory principles for a large number of biological processes. We show that different regulatory patterns between functionally similar proteins are characterized by differences in the half-life of the corresponding transcripts and can be identified by measuring RNA half-life. We identify more than 100 protein families which show such differential regulatory patterns in both species. Additionally, we provide strong evidence that the activity of protein complexes consisting of subunits with overall long transcript half-lives can be regulated by transcriptional regulation of individual key subunits with short-lived transcripts. Based on this observation, we predict more than 100 key regulatory subunits for human complexes of which 28% could be confirmed in mice (P < 10(-9)). Therefore, this atlas of transcript half-lives provides new fundamental insights into many cellular processes.
Prostate-specific membrane antigen (PSMA) is an attractive target for the diagnosis and therapy of prostate cancer as it is specifically overexpressed in prostate cancer cells. Improving the circulation of radioligands in the blood is considered as an effective strategy that can improve tumor burden, which benefits detection of small lesions and improves the effect of PSMA radioligand therapy (PRLT). In this study, we introduced maleimidopropionic acid (MPA) to a PSMA-targeted tracer and developed Al18F-PSMA-CM, which targets human serum albumin (HSA) binding and PSMA. Al18F-PSMA-CM is evaluated in vitro and in vivo for stability, PSMA specificity, and biodistribution in 22Rv1 tumor-bearing mice. Al18F-PSMA-CM was prepared with a radiochemical purity of >99% and specific activity of 11.22-18.70 MBq/nmol. Al18F-PSMA-CM was stable in vitro and in vivo and prolonged circulation in blood with a binding ratio of 47 ± 3.2% and Kd value of 3.08 ± 0.45 nM to HSA. The uptake of Al18F-PSMA-CM in PSMA(+) 22Rv1 cells was increased in 2 h, and the uptake was blocked by a PSMA inhibitor, ZJ-43. The Kd value of Al18F-PSMA-CM to PSMA was 8.46 ± 0.24 nM. Al18F-PSMA-CM was accumulated in kidneys and 22Rv1 tumors [74.76 ± 15.42 and 6.16 ± 0.74 ID%/g at 2 h post injection (p.i.)], which were decreased by -80.0 and -84.3% when co-injected with ZJ-43. Al18F-PSMA-CM showed high PSMA specificity and accumulated in 22Rv1 tumors with increasing uptake in 4 h. MPA moiety showed the ability to prolong the half-life of tracers, and the MPA-conjugated tracer showed the potential to improve tumor uptake. MPA may be a choice to develop radiopharmaceuticals for PRLT of prostate cancer.
Standard clinical X-ray contrast agents are small iodine-containing molecules that are rapidly cleared by the kidneys and provide robust imaging for only a few seconds, thereby limiting more extensive vascular and tissue biodistribution imaging as well as optimal tumor uptake. They are also not generally useful for preclinical microCT imaging where longer scan times are required for high resolution image acquisition. We here describe a new iodine nanoparticle contrast agent that has a unique combination of properties: 20 nm hydrodynamic diameter, covalent PEG coating, 40 hour blood half-life, 50% liver clearance after six months, accumulation in tumors, and well-tolerated to at least 4 g iodine/kg body weight after intravenous administration in mice. These characteristics are unique among the other iodine nanoparticles that have been previously reported and provide extended-time high contrast vascular imaging and tumor loading. As such, it is useful for preclinical MicroCT animal studies. Potential human applications might include X-ray radiation dose enhancement for cancer therapy and vascular imaging for life-threatening situations where high levels of contrast are needed for extended periods of time.
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