Proteome Profile of Endogenous Retrotransposon‐Associated Complexes in Human Embryonic Stem Cells

Vuong, Linh; Pan, Songqin; Donovan, Peter

doi:10.1002/pmic.201900169

Cited by 9 publications

(11 citation statements)

References 37 publications

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“…We also tried to IP ORF2p from CRC tissues and PA-1 cells using anti-ORF2p antibodies, followed by western blotting against ORF2p, but were unable to detect ORF2p (not shown); further optimizations may alter this outcome. While preparing this manuscript, a dataset was released comprising anti-ORF1p co-IPs from H9 human embryonic stem cells with LFQ MS-based analysis [65], which have been shown to exhibit strong ORF1p expression [66]. ORF2p detection was not reported.…”

Section: Discussionmentioning

confidence: 99%

LINE-1 ORF2p expression is nearly imperceptible in human cancers

et al. 2019

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BackgroundLong interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines.ResultsWe report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743.ConclusionsAlthough somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

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Section: Discussionmentioning

confidence: 99%

LINE-1 ORF2p expression is nearly imperceptible in human cancers

et al. 2019

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“…We also tried to IP ORF2p from CRC tissues and PA-1 cells using anti-ORF2 antibodies, followed by western blotting against ORF2p, but were unable to detect ORF2p (not shown); further optimizations may alter this outcome. While preparing this manuscript, a dataset was released comprising anti-ORF1p co-IPs from H9 human embryonic stem cells with LFQ MS-based analysis [58]; ORF2p detection was not reported. ORF2p peptide detection has previously been reported by MALDI-TOF MS after IP with an anti-ORF2p polyclonal antibody [59]; however, this reagent no longer exists so, further validation and side-by-side comparisons are not possible.…”

Section: Discussionmentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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Background: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results:We report mass spectrometry based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically-acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions:Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth. References

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“…However, studies have shown that chA1-L1 recognizes both ORF2p and the transcriptional regulator BCLAF1, so it is not specific (Briggs et al, 2019). But recently, tumor proteome profiling studies based on mass spectrometry have shown that ORF2p was difficult to be detected, and after affinity capture of ORF1p, ORF2p has not been detected in stem cell LINE-1 proteome analysis (Vuong et al, 2019;Ardeljan et al, 2020). Therefore, the detection and application of ORF2 in tumors are still worth exploring.…”

Section: High Expression Of Orf1 and Orf2 Of Line-1 And Cancermentioning

confidence: 99%

New Understanding of the Relevant Role of LINE-1 Retrotransposition in Human Disease and Immune Modulation

Zhang

2020

Front. Cell Dev. Biol.

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Long interspersed nuclear element-1 (LINE-1) retrotransposition is a major hallmark of cancer accompanied by global chromosomal instability, genomic instability, and genetic heterogeneity and has become one indicator for the occurrence, development, and poor prognosis of many diseases. LINE-1 also modulates the immune system and affects the immune microenvironment in a variety of ways. Aberrant expression of LINE-1 retrotransposon can provide strong stimuli for an innate immune response, activate the immune system, and induce autoimmunity and inflammation. Therefore, inhibition the activity of LINE-1 has become a potential treatment strategy for various diseases. In this review, we discussed the components and regulatory mechanisms involved with LINE-1, its correlations with disease and immunity, and multiple inhibitors of LINE-1, providing a new understanding of LINE-1.

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Proteome Profile of Endogenous Retrotransposon‐Associated Complexes in Human Embryonic Stem Cells

Cited by 9 publications

References 37 publications

LINE-1 ORF2p expression is nearly imperceptible in human cancers

LINE-1 ORF2p expression is nearly imperceptible in human cancers

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

New Understanding of the Relevant Role of LINE-1 Retrotransposition in Human Disease and Immune Modulation

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