mCherry contains a fluorescent protein isoform that interferes with its reporter function

Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion

Background. The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results. Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen ( p ≤ 0.05 ). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions. Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

Effect of TMEM154 E35K variant (haplotypes “1” and “3”) on the incidence of ovine lentivirus infection and ewe productivity during lifetime exposure

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus that is widespread throughout U.S. sheep flocks. Infections with OPPV are lifelong and effects are multi-systemic with significant implications for animal well-being and productivity. A protein isoform with lysine at position 35 (K35, haplotype “1”) encoded by the ovine transmembrane protein 154 (TMEM154) gene has been associated with reduced susceptibility to infection when two copies are present (i.e., diplotype “1,1”). Conversely, the ancestral protein isoform with glutamate at position 35 (E35, haplotype “3”) is associated with high susceptibility to infection when at least one copy is present. The beneficial effect of TMEM154 K35 alleles on ewe productivity has not been previously measured in controlled challenge experiments and was a major objective of this study. Ewes with TMEM154 diplotypes “1,1”; “1,3”; and “3,3” (n = 31, 47, and 30, respectively) were born and reared by OPPV-infected dams and managed under continual natural exposure to OPPV. Ewes were tested for serological status at 4 mo intervals for up to 5.5 yr. The incidence of infection in ewes with diplotype “1,1” was 6.5 to 9.7% and significantly lower (P < 0.001) than ewes with diplotype “1,3” (60.5 to 97.3%) or “3,3” (64.0 to 91.4%). Furthermore, the incidence among ewes with diplotype “1,1” did not increase from 10 to 67 mo of age (P > 0.99), whereas the incidence among diplotype “1,3” and “3,3” ewes increased steadily until reaching an asymptote at approximately 52 mo of age. Total number and weight of lamb weaned per ewe exposed through 5.5 yr from ewes with diplotype “1,1” far exceeded (P ≤ 0.05) those with diplotypes “1,3” and “3,3” by, on average, 2.1 lambs and 40 kg, respectively. The present study confirmed that TMEM154 diplotype “1,1” animals have reduced incidence of OPPV infection and, correspondingly, improved productivity. In flocks with a high frequency of TMEM154 haplotype “3”, selection for haplotype “1” appears to be a cost-effective approach to mitigate the impact of this economically important disease.

Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress

The emergence of new primate-specific genes is an essential factor in human and primate brain development and functioning. POU2F1/Oct-1 is a transcription regulator in higher eukaryotes which is involved in the regulation of development, differentiation, stress response, and other processes. We have demonstrated that the Tigger2 transposon insertion into the POU2F1 gene which occurred in the primate lineage led to the formation of an additional exon (designated the Z-exon). Z-exon-containing primate-specific Oct-1Z transcript includes a short upstream ORF (uORF) located at its 5’-end and the main ORF encoding the Oct-1Z protein isoform (Pou2F1 isoform 3, P14859-3), which differs from other Oct-1 isoforms by its N-terminal peptide. The Oct-1Z-encoding transcript is expressed mainly in human brain cortex. Under normal conditions, the translation of the ORF coding for the Oct-1Z isoform is repressed by uORF. Under various stress conditions, uORF enables a strong increase in the translation of the Oct-1Z-encoding ORF. Increased Oct-1Z expression levels in differentiating human neuroblasts activate genes controlling stress response, neural cell differentiation, brain formation, and organogenesis. We have shown that the Oct-1Z isoform of the POU2F1/Oct-1 transcription factor is an example of a primate-specific genomic element contributing to brain development and cellular stress defense.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML.

NMRK2 Gene Is Upregulated in Dilated Cardiomyopathy and Required for Cardiac Function and NAD Levels during Aging

Dilated cardiomyopathy (DCM) is a disease of multifactorial etiologies, the risk of which is increased by male sex and age. There are few therapeutic options for patients with DCM who would benefit from identification of common targetable pathways. We used bioinformatics to identify the Nmrk2 gene involved in nicotinamide adenine dinucleotde (NAD) coenzyme biosynthesis as activated in different mouse models and in hearts of human patients with DCM while the Nampt gene controlling a parallel pathway is repressed. A short NMRK2 protein isoform is also known as muscle integrin binding protein (MIBP) binding the α7β1 integrin complex. We investigated the cardiac phenotype of Nmrk2-KO mice to establish its role in cardiac remodeling and function. Young Nmrk2-KO mice developed an eccentric type of cardiac hypertrophy in response to pressure overload rather than the concentric hypertrophy observed in controls. Nmrk2-KO mice developed a progressive DCM-like phenotype with aging, associating eccentric remodeling of the left ventricle and a decline in ejection fraction and showed a reduction in myocardial NAD levels at 24 months. In agreement with involvement of NMRK2 in integrin signaling, we observed a defect in laminin deposition in the basal lamina of cardiomyocytes leading to increased fibrosis at middle age. The α7 integrin was repressed at both transcript and protein level at 24 months. Nmrk2 gene is required to preserve cardiac structure and function, and becomes an important component of the NAD biosynthetic pathways during aging. Molecular characterization of compounds modulating this pathway may have therapeutic potential.

Transcriptional Regulation of Postnatal Cardiomyocyte Maturation and Regeneration

During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the heart, contributing to heart failure after cardiac injury in adults. While most studies focus on the transcriptional regulation of embryonic or adult cardiomyocytes, the transcriptional changes that occur during the postnatal period are relatively unknown. In this review, we focus on the transcriptional regulators responsible for these aspects of cardiomyocyte maturation during the postnatal period in mammals. By specifically highlighting this transitional period, we draw attention to critical processes in cardiomyocyte maturation with potential therapeutic implications in cardiovascular disease.