In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

<div><p>The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.</p> </div

Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library.

<p>(<b>A</b>) Western blot analysis of N-terminal YFP1-tagged fusion proteins expressed by the constructs with and without 5′-UTRs in HEK-293T cells. Expression of a construct containing only YFP1 was used as a control. An anti-GFP N-terminal antibody was used to visualize the expressed tagged proteins. (<b>B</b>) YFP fluorescence in HEK-293T cells after the co-transfection of YFP1-<i>CRABP2</i> with <i>ARL11</i>-YFP2 and YFP1-<i>PGAM1</i> with <i>ARL11</i>-YFP2. Nuclei were counterstained with DAPI. (<b>C</b>) Confirmation of the interaction between ARL11 and CRABP2 by western blotting and co-immunoprecipitation. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and FLAG-tagged <i>CRABP2</i> constructs without its 5′-UTR. Protein expression was verified by immunoblotting using anti-ARL11 and anti-CRABP2 antibodies in direct western blots (DWB). Immunoprecipitation with western blotting (IPWB) was performed by anti-HA antibody pull-down of ARL11 to detect CRABP2 binding (top panel). Results were confirmed using a complementary approach (HA-tagged CRABP2, anti-HA antibody immunoprecipitation, and anti-ARL11 immunoblotting (bottom panel). (<b>D</b>) Confirmation of ARL 11 and PGAM1 binding by IPWB. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and flag-tagged <i>PGAM1</i> constructs as indicated. Protein expression verified by immunoblotting with anti-PGAM1 (top panel) or anti-ARL11 (bottom panel) antibodies (DWBs). IPWBs were performed by anti-HA immunoprecipiation of ARL11 followed by immunoblotting with anti-PGAM1 (Top panel). Alternatively, immunoprecipitation was performed using HA-tagged PGAM1 followed by immunoblotting with anti-ARL11 (Bottom panel). (<b>E</b>) The in-frame cDNA library prevented interference caused by the <i>CRABP2</i> 5′-UTR that inhibits its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and with YFP1-<i>CRABP2</i> or YFP1-5′-UTR-<i>CRABP2</i> as indicated. Protein expression was confirmed by immunoblotting with anti-CRABP2 antibody (top panel) or anti-YFP1 antibody (bottom panel). Alternatively, ARL11 was immunoprecipitated using the anti-HA antibody, and bound proteins were detected by immunoblotting with anti-CRABP2 (top panel) or anti-YFP1 (bottom panel) antibody. (<b>F</b>) The in-frame cDNA library prevents interference caused by the <i>PGAM1</i> 5′-UTR that prevents its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and YFP1-<i>PGAM1</i>, or YFP1-5′-UTR-<i>PGAM1</i>. Protein expression was confirmed using anti-PGAM1 (top panel) or anti-YFP1 (bottom panel) antibodies (DWB). To identify ARL11-associated proteins (IPWB), ARL11 was immunoprecipitated using the anti-HA antibody and bound proteins were detected using either an anti-PGM1 (top panel) or anti-YFP (bottom panel) antibody.</p

Predicted expression of CRABP2 and PGAM1 proteins by the constructs with and without 5′-UTRs.

<p>(<b>A</b>) Comparison of <i>CRABP2</i> expression constructs with and without the <i>CRABP2</i> 5′-UTR. (<b>B</b>) Comparison of <i>PGAM1</i> expression constructs with and without the <i>PGAM1</i> 5′-UTR.</p

Analysis of the human 5′-UTR database, overview of the approach, and construction of the in-frame cDNA expression library.

<p>(<b>A</b>) Analysis of the human 5′-UTR database (<a href="http://utrdb.ba.itb.cnr.it/" target="_blank">http://utrdb.ba.itb.cnr.it/</a>) to predict their effects on expressed sequences following translation with a YFP1 tag peptide as fusion proteins during the construction of a prey cDNA library. (<b>B</b>) Overview of the screening procedure. (<b>C</b>) For the construction of the in-frame cDNA expression library, mRNA was isolated from normal human urothelial cells and was used as a template for first-strand cDNA synthesis using polyT primer. Double-stranded cDNAs without 5′-UTRs were synthesized using primers 1 and 2 (representing approximately 40% of the Kozak sequences that are present in vertebrate genomes) complemented with primer mixes 3 and 4 (representing the remaining 60% of the Kozak sequence combinations in vertebrates). In primer mixes 3 and 4, the combination of sequences referred to as “D” is an equal mixture of A, G and T, “H” is an equal mixture of A, C and T, “K” is an equal mixture of G and T, and “W” is an equal mixture of A and T. There are 19,683 and 157,464 possible sequence combinations in primer mixes 3 and 4, respectively. (<b>D</b>) Sequence analysis of the in-frame cDNA library was performed on 198 representative plasmids isolated from random colonies of the library.</p

Prediction of a Multi-Gene Assay (Oncotype DX and Mammaprint) Recurrence Risk Group Using Machine Learning in Estrogen Receptor-Positive, HER2-Negative Breast Cancer—The BRAIN Study

This study aimed to develop a machine learning-based prediction model for predicting multi-gene assay (MGA) risk categories. Patients with estrogen receptor-positive (ER+)/HER2− breast cancer who had undergone Oncotype DX (ODX) or MammaPrint (MMP) were used to develop the prediction model. The development cohort consisted of a total of 2565 patients including 2039 patients tested with ODX and 526 patients tested with MMP. The MMP risk prediction model utilized a single XGBoost model, and the ODX risk prediction model utilized combined LightGBM, CatBoost, and XGBoost models through soft voting. Additionally, the ensemble (MMP + ODX) model combining MMP and ODX utilized CatBoost and XGBoost through soft voting. Ten random samples, corresponding to 10% of the modeling dataset, were extracted, and cross-validation was performed to evaluate the accuracy on each validation set. The accuracy of our predictive models was 84.8% for MMP, 87.9% for ODX, and 86.8% for the ensemble model. In the ensemble cohort, the sensitivity, specificity, and precision for predicting the low-risk category were 0.91, 0.66, and 0.92, respectively. The prediction accuracy exceeded 90% in several subgroups, with the highest prediction accuracy of 95.7% in the subgroup that met Ki-67 <20 and HG 1~2 and premenopausal status. Our machine learning-based predictive model has the potential to complement existing MGAs in ER+/HER2− breast cancer

Intermediate cells of in vitro cellular reprogramming and in vivo tissue regeneration require desmoplakin

Amphibians and fish show considerable regeneration potential via dedifferentiation of somatic cells into blastemal cells. In terms of dedifferentiation, in vitro cellular reprogramming has been proposed to share common processes with in vivo tissue regeneration, although the details are elusive. Here, we identified the cytoskeletal linker protein desmoplakin (Dsp) as a common factor mediating both reprogramming and regeneration. Our analysis revealed that Dsp expression is elevated in distinct intermediate cells during in vitro reprogramming. Knockdown of Dsp impedes in vitro reprogramming into induced pluripotent stem cells and induced neural stem/progenitor cells as well as in vivo regeneration of zebrafish fins. Notably, reduced Dsp expression impairs formation of the intermediate cells during cellular reprogramming and tissue regeneration. These findings suggest that there is a Dsp-mediated evolutionary link between cellular reprogramming in mammals and tissue regeneration in lower vertebrates and that the intermediate cells may provide alternative approaches for mammalian regenerative therapy.11Nsciescopu