The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFB binding sequences and monitoring tumor necrosis factor ␣-mediated NFB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFB translocation to the nucleus.Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization (1-4). GFP fluorescence does not require any substrate or cofactor (5); hence it is possible to use it in many species for live cell detection purposes. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65-67). This sequence undergoes spontaneous oxidation to form a cyclized chromophore (6). Enhanced GFP (EGFP) contains mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with humanoptimized codons (7-9). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the GFP tertiary structure resembles a barrel (10, 11). GFP is a single chain polypeptide of 238 amino acids (12). Most of these amino acids form  sheets that are compacted through an antiparallel structure to form the barrel. An ␣-helix containing the chromophore is located inside the barrel, which shields it from the external environment. The compact structure makes GFP very stable under a variety of conditions, including treatment with protease (1). The stability of GFP limits its application in some studies, including transcriptional induction studies.Cellular proteins differ widely in their stabilities. Rapid turnover in proteins is often caused by signals that induce protein degradation. In some cases, the signal is a primary sequence such as the PEST sequence, a sequence possibly correlated with protein degradation (13,14)...
Regulated degradation of specific proteins is part of the intracellular biochemical changes that contribute to the regulation of signal transduction pathways, cell proliferation, growth arrest, and apoptosis. For instance, tumor necrosis factor (TNF) 1 ␣-mediated NF B activation requires the rapid degradation of I B␣, the inhibitor of NF B (1-4). NF B is a transcription factor that regulates the expression of a number of genes whose products contribute to inflammation and immune responses (5, 6). Before activation, NF B is sequestered in the cytoplasm by forming a complex with I B␣ (7-9). The nuclear translocation signal of NF B is masked by the inhibitor (10). NF B can be activated by a number of stimuli, including TNF␣, interleukin 1, lipopolysaccharide, and phorbol esters (PMA). Initiated by TNF␣ binding, a number of proteins including TRADD, TRAF2, and RIP aggregate around the TNF␣ type 1 receptor (11-13), which triggers the phosphorylation of I B␣ and leads to the rapid dissociation of NF B from I B␣ (14 -17). A protein kinase complex, which includes NIK, IKK␣, and IKK, is involved in the phosphorylation of I B␣ (18 -22). The phosphorylated I B␣ is further modified by ubiquitination enzymes and degraded by the 26S proteasome (23). Serine residues 32 and 36 of I B␣ have been identified to be the specific target sites of phosphorylation (16, 24 -26). Mutations at these positions abolish both phosphorylation and degradation of I B␣ (16, 24 -26). Once released from the I B␣ complex, NF B immediately translocates from the cytoplasm to the nucleus, where it mediates the transcriptional activation of genes, such as interleukin 2 and I B␣. I B␣ degradation is rapid and is completed within 5-40 min after stimulation by TNF␣ (1, 2, 7). I B␣ also degrades in the absence of TNF␣, but this basal degradation is much slower than induced degradation. The half-life of the basal degradation is around 2 h (2, 29). It is unclear whether the basal degradation of I B␣ also requires phosphorylation for initiation of the degradation process (30). Therefore, the relationship of basal degradation to induced degradation remains uncertain.Both the gene and cDNA of the green fluorescent protein (GFP) have been cloned from the jellyfish Aequorea victoria (31). GFP has been widely used to study gene expression and protein localization (32-35) because its fluorescence emission does not require substrates or cofactors (36), and fluorescence detection can be made in real time. The key sequence of SerTyr-Gly (amino acids 65-67) within GFP undergoes spontaneous oxidation to form a cyclized chromophore that emits fluorescence (37). Mutation of Ser to Thr in the chromophore (S65T) leads to a higher fluorescence intensity of GFP. Enhanced GFP (EGFP) is one such mutant. It contains the mutations S65T and F64L and is encoded by a gene with humanoptimized codons (38 -40). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the tertiary structure of GFP resembles a barrel (41, 42), and this compact structure makes GFP a ver...
Vertical transmission of SARS-CoV-2, the virus responsible for COVID-19, from parents to early embryos during conception could be catastrophic, but is contingent on the susceptibility of cells of the embryo to infection. Because presence of the SARS-CoV-2 virus has been reported in the human reproductive system, we assessed whether pre-implantation embryos are permissive to SARS-CoV-2 entry. RNA-seq and immunostaining studies revealed presence of two key entry factors in the trophectoderm of blastocyst-stage embryos, the ACE2 receptor and the TMPRSS2 protease. Exposure of blastocysts to fluorescent reporter virions pseudotyped with the SARS-CoV-2 Spike (S) glycoprotein revealed S-ACE2 dependent entry and fusion. These results indicate that human pre-implantation embryos can be infected by SARS-CoV-2, a finding pertinent to natural human conceptions and assisted reproductive technologies during and after the COVID-19 pandemic.
The spread of SARS-CoV-2 has led to a devastating pandemic, with infections resulting in a range of symptoms collectively known as COVID-19. The full repertoire of human tissues and organs susceptible to infection is an area of active investigation, and some studies have implicated the reproductive system. The effects of COVID-19 on human reproduction remain poorly understood, and particularly the impact on early embryogenesis and establishment of a pregnancy are not known. In this work, we explore the susceptibility of early human embryos to SARS-CoV-2 infection. By using RNA-seq and immunofluorescence, we note that ACE2 and TMPRSS2, two canonical cell entry factors for SARS-CoV-2, are co-expressed in cells of the trophectoderm in blastocyst-stage preimplantation embryos. For the purpose of viral entry studies, we used fluorescent reporter virions pseudotyped with Spike (S) glycoprotein from SARS-CoV-2, and we observe robust infection of trophectoderm cells. This permissiveness could be attenuated with blocking antibodies targeting S or ACE2. When exposing human blastocysts to the live, fully infectious SARS-CoV-2, we detected cases of infection that compromised embryo health. Therefore, we identify a new human target tissue for SARS-CoV-2 with potential medical implications for reproductive health during the COVID-19 pandemic and its aftermath.
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