Expression Of Protein Isoforms Research Articles

Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease

A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N= ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs in TPM2, in which siRNA-mediated knockdown in osteoblasts showed two TPM2 isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.

IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

Emerging concepts of myosin 18A isoform mechanobiology in organismal and immune system physiology, development, and function.

Alternative and combinatorial splicing of myosin 18A (MYO18A) gene transcripts results in expression of MYO18A protein isoforms and isoform variants with different membrane and subcellular localizations, and functional properties. MYO18A proteins are members of the myosin superfamily consisting of a myosin-like motor domain, an IQ motif, and a coiled-coil domain. MYO18A isoforms, however, lack the ability to hydrolyze ATP and do not perform ATP-dependent motor activity. MYO18A isoforms are distinguished by different amino- and carboxy-terminal extensions and domains. The domain organization and functions of MYO18Aα, MYO18Aβ, and MYO18Aγ have been studied experimentally. MYO18Aα and MYO18Aβ have a common carboxy-terminal extension but differ by the presence or absence of an amino-terminal KE repeat and PDZ domain, respectively. The amino- and carboxy-terminal extensions of MYO18Aγ contain unique proline and serine-rich domains. Computationally predicted MYO18Aε and MYO18Aδ isoforms contain the carboxy-terminal serine-rich extension but differ by the presence or absence of the amino-terminal KE/PDZ extension. Additional isoform variants within each category arise by alternative utilization or inclusion/exclusion of small exons. MYO18Aα variants are expressed in somatic cells and mature immune cells, whereas MYO18Aβ variants occur mainly in myeloid and natural killer cells. MYO18Aγ expression is selective to cardiac and skeletal muscle. In the present review perspective, we discuss current and emerging concepts of the functional specialization of MYO18A proteins in membrane and cytoskeletal dynamics, cellular communication and signaling, endocytic and exocytic organelle movement, viral infection, and as the SP-R210 receptor for surfactant protein A.

GLUT2 regulation of p38 MAPK isoform protein expression and p38 phosphorylation in male versus female rat hypothalamic primary astrocyte Cultures

Recent studies documented regulation of hypothalamic astrocyte mitogen-activated protein kinase (MAPK) pathways, including p38, by the plasma membrane glucose carrier/sensor glucose transporter-2 (GLUT2). Sex-specific GLUT2 control of p38 phosphorylation was observed, but effects on individual p38 family protein profiles were not investigated. Current research employed an established primary astrocyte culture model, gene knockdown tools, and selective primary antisera against p38-alpha, p38-beta, p38-gamma, and p38-delta isoforms to investigate whether GLUT2 governs expression of one or more of these variants in a glucose-dependent manner. Data show that GLUT2 inhibits baseline expression of each p38 protein in male cultures, yet stimulates p38-delta profiles without affecting other p38 proteins in female. Glucose starvation caused selective up-regulation of p38-delta profiles in male versus p38-alpha and -gamma proteins in female; these positive responses were amplified by GLUT2 siRNA pretreatment. GLUT2 opposes or enhances basal p38 phosphorylation in male versus female, respectively. GLUT2 siRNA pretreatment did not affect glucoprivic patterns of phospho-p38 protein expression in either sex. Outcomes document co-expression of the four principal p38 MAPK family proteins in hypothalamic astrocytes, and implicate GLUT2 in regulation of all (male) versus one (female) variant(s). Glucoprivation up-regulated expression of distinctive p38 isoforms in each sex; these stimulatory responses are evidently blunted by GLUT2. Glucoprivic-associated loss of GLUT2 gene silencing effects on p38 phosphorylation infers either that glucose status determines whether this sensor controls phosphorylation, or that decrements in screened glucose in each instance are of sufficient magnitude to abolish GLUT2 regulation of that function.

Effects of dietary nitrogen and/or phosphorus reduction on mineral homeostasis and regulatory mechanisms in young goats.

The reduction of nitrogen (N) and phosphorus (P) in ruminant feed is desirable due to costs and negative environmental impact. Ruminants are able to utilize N and P through endogenous recycling, particularly in times of scarcity. When N and/or P were reduced, changes in mineral homeostasis associated with modulation of renal calcitriol metabolism occurred. The aim of this study was to investigate the potential effects of dietary N- and/or P-reduction on the regulatory mechanisms of mineral transport in the kidney and its hormonal regulation in young goats. During N-reduction, calcium (Ca) and magnesium (Mg) concentrations in blood decreased, accompanied by a lower protein expression of cytochrome P450 family 27 subfamily B member 1 (CYP27B1) (p = 0.016). The P-reduced fed goats had low blood phosphate concentrations with simultaneously high Ca and Mg levels. The insulin-like growth factor 1 concentrations decreased significantly with P-reduction. Furthermore, gene expression of CYP27B1 (p < 0.001) and both gene (p = 0.025) and protein (p = 0.016) expression of the fibroblast growth factor receptor 1c isoform in the kidney were also significantly reduced during a P-reduced diet. ERK1/2 activation exhibited a trend toward reduction in P-reduced animals. Interestingly, calcitriol concentrations remained unaffected by either restriction individually, but interacted significantly with N and P (p = 0.014). Additionally, fibroblast growth factor 23 mRNA expression in bone decreased significantly with P-restriction (p < 0.001). These results shed light on the complex metabolic and regulatory responses of mineral transport of young goats to dietary N and P restriction.

Na/K-ATPase α1/Src Signaling Regulates Mitochondrial Metabolic Function and ROS Production in Human iPSC-derived Cardiomyocytes

Through an isoform specific mechanism, the Na/K-ATPase (NKA) α1 polypeptide-mediated regulation of Src kinase has emerged as a novel regulator of mitochondrial function in various cell types. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To investigate the importance of the proposed NKA/Src regulatory signaling on cardiac mitochondrial metabolic function independently from the classic Na/K-ATPase enzymatic function, we used a gene targeting approach in cardiomyocytes. Human induced pluripotent stem cells (hiPSC) expressing a Src-signaling null mutant (A420P) form of NKA α1 were generated using CRISPR/Cas9-mediated genome editing. NKA isoform protein expression and enzymatic activity remained unchanged. However, baseline levels of Src and ERK1/2 phosphorylation were decreased by 55% and 80%, respectively in the A420P hiPSC (n=3, p&lt;0.05). Both the WT and A420P hiPSC differentiated into cardiomyocytes (iCM) as assessed by morphology, spontaneous cell contraction, marker gene expression, and subcellular striations. Cell metabolism assessed by Seahorse extracellular flux analysis revealed significant reductions in basal and maximal rates of mitochondrial respiration (50%), spare respiratory capacity (50%), ATP production (60%), and coupling effciency (20%) (n=4, p&lt;0.05). ROS production was monitored in live cells by measuring the oxidation of the CM-H2DCFDA dye using a fluorescence microscope. The slope of the ROS production rate curve was dramatically reduced in A420P iCM (80%, n=3, p&lt;0.05). Protein carbonylation levels, analyzed by ELISA as markers of oxidative damage, were also decreased in A420P iCM (n=4, p&lt;0.05). Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This has potential significance in cardiometabolic diseases and suggests that targeting the NKA α1/Src signaling axis may offer a novel approach to address mitochondrial dysfunction. NIH Grant R15 HL145666, NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence, and American Heart Association Postdoctoral Fellowship — 22POST917776 — to Marco T. Pessoa. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration.

Oxidative stress represents a hallmark for many degenerative pathologies of the Central Nervous System. Throughout life, the constant pressure of noxious stimuli and/or episodes of traumatic events may expose the brain to a microenvironment where the non-balanced reactive oxygen species inevitably lead to neuronal loss and cognitive decline. HO-1, a 32 kDa heat-shock protein catalyzing the degradation of heme into carbon monoxide (CO), iron and biliverdin/bilirubin is considered one of the main antioxidant defense mechanisms playing pivotal roles in neuroprotection. Restoring the redox homeostasis is the goal of many natural or synthetic antioxidant molecules pursuing beneficial effects on brain functions. Here, we investigated the antioxidant capacity of four selected benzofuran-2-one derivatives in a cellular model of neurodegeneration represented by differentiated SH-SY5Y cells exposed to catechol-induced oxidative stress. Our main results highlight how all the molecules have antioxidant properties, especially compound 9, showing great abilities in reducing intracellular ROS levels and protecting differentiated SH-SY5Y cells from catechol-induced death. This compound above all seems to boost HO-1 mRNA and perinuclear HO-1 protein isoform expression when cells are exposed to the oxidative insult. Our findings open the way to consider benzofuran-2-ones as a novel and promising adjuvant antioxidant strategy for many neurodegenerative disorders.

Neuron type-specific proteomics reveals distinct Shank3 proteoforms in iSPNs and dSPNs lead to striatal synaptopathy in Shank3B-/- mice.

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies. To overcome the limitations associated with bulk measures of synaptic protein abundance, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Investigation of Shank3B-/- mice lacking exons 13-16 within the Shank3 gene, reveal distinct Shank3 protein isoform expression in iSPNs and dSPNs. In Shank3B-/- striatum, Shank3E and Shank3NT are expressed by dSPNs but are undetectable in iSPNs. Proteomic analysis indicates significant and selective alterations in the postsynaptic proteome of Shank3B-/- iSPNs. Correspondingly, the deletion of exons 13-16 diminishes dendritic spine density, reduces spine head diameter, and hampers corticostriatal synaptic transmission in iSPNs. Remarkably, reintroducing Shank3E in adult Shank3B-/- iSPNs significantly rectifies the observed dendritic spine morphological and corticostriatal synaptic transmission deficits. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

The Muscleblind-like protein MBL-1 regulates microRNA expression in Caenorhabditis elegans through an evolutionarily conserved autoregulatory mechanism.

The Muscleblind-like (MBNL) family is a highly conserved set of RNA-binding proteins (RBPs) that regulate RNA metabolism during the differentiation of various animal tissues. Functional insufficiency of MBNL affects muscle and central nervous system development, and contributes to the myotonic dystrophies (DM), a set of incurable multisystemic disorders. Studies on the regulation of MBNL genes are essential to provide insight into the gene regulatory networks controlled by MBNL proteins and to understand how dysregulation within these networks causes disease. In this study, we demonstrate the evolutionary conservation of an autoregulatory mechanism that governs the function of MBNL proteins by generating two distinct protein isoform types through alternative splicing. Our aim was to further our understanding of the regulatory principles that underlie this conserved feedback loop in a whole-organismal context, and to address the biological significance of the respective isoforms. Using an alternative splicing reporter, our studies show that, during development of the Caenorhabditis elegans central nervous system, the orthologous mbl-1 gene shifts production from long protein isoforms that localize to the nucleus to short isoforms that also localize to the cytoplasm. Using isoform-specific CRISPR/Cas9-generated strains, we showed that expression of short MBL-1 protein isoforms is required for healthy neuromuscular function and neurodevelopment, while expression of long MBL-1 protein isoforms is dispensable, emphasizing a key role for cytoplasmic functionalities of the MBL-1 protein. Furthermore, RNA-seq and lifespan analyses indicated that short MBL-1 isoforms are crucial regulators of miRNA expression and, in consequence, required for normal lifespan. In conclusion, this study provides support for the disruption of cytoplasmic RNA metabolism as a contributor in myotonic dystrophy and paves the way for further exploration of miRNA regulation through MBNL proteins during development and in disease models.

Abstract C124: Assessment of the anticancer potential of a natural compound euphol in colorectal cancer from in vitro and in vivo mouse models

Abstract Introduction and objectives: Euphol, a tetracyclic triterpene alcohol, is the main component in the sap of the Euphorbiaceae family of plants in subtropical and tropical regions. It has been reported as a modulator of protein kinases C (PKC) activity and to possess several properties, including anti-inflammatory and, more recently, antineoplastic action. These findings further amplified the therapeutic interest in euphol for cancer prevention and treatment. In the present study, we aimed to explore the in vitro and in vivo antitumor effect of synthetic euphol on colorectal cancer (CRC) to provide insight into euphol-based therapies for cancer patients. Methods: Cytotoxicity potential of commercial synthetic euphol on CRC cell lines (HCT15, LOVO – MSI) and (SW620, HT29 – MSS) and mouse organoids derived from ApcLoxP/+-Cdx2 mice was analyzed by MTS-based viability assay using CellTiterGlo kit. The clonogenic potential of CRC cell lines was assessed following a 10-day treatment with different doses of euphol. Quantitative RT-PCR and Western blot analyses were performed to study the effects of euphol on the gene and protein expression of the PKCs isoforms in CRC cell lines and organoids. In addition, a PDX mouse model was established using a surgical sample from a CRC patient. For in vivo studies, the PDX model and ApcLoxP/+-Cdx2 mice were administered euphol or vehicle by mouth for 10 days to assess the therapeutic effect and 8 weeks to assess the preventive effect, respectively. Results: Treatment with euphol resulted in reduced cell viability in both MSS and MSI CRC cell lines, and mouse organoids, following a 72-hour exposure. Euphol also promoted the reduction of the clonogenic potential of CRC cell lines in a dose-dependent manner. Further, the PKCα, PKCβ, PKCδ, and PKCZ gene and protein expression were stimulated in euphol-treated organoids, compared to control. Notably, we observed a significant decrease in tumor volume and suppressed tumor growth after 10 days of treatment with euphol in a PDX mouse model. Euphol-treated ApcLoxP/+-Cdx2 mice for 8 weeks also showed reduced colonic polyposis compared to untreated mice. Conclusions: These results indicate that synthetic euphol has a cytotoxic potential in inhibiting cell proliferation of CRC cells in both 2D and 3D models, and in vivo models through modulation of the expression of genes involved PKC pathway, thus highlighting the therapeutic potential of euphol for CRC. Citation Format: Ana Laura Vieira Alves, Ana Carolina Martin, Krishna M Sinha, Viviane Aline Oliveira Silva, Renato José da Silva-Oliveira, Silvia A Teixeira, Denise P Guimaraes, Luis Gustavo Romagnolo, Monise Tadin Reis, Fahriye Duzagac, Eduardo Vilar, Rui Manuel Reis. Assessment of the anticancer potential of a natural compound euphol in colorectal cancer from in vitro and in vivo mouse models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C124.

Exercise training and vascular heterogeneity in db/db mice: evidence for regional- and duration-dependent effects.

Exercise training (ET) has several health benefits; however, our understanding of regional adaptations to ET is limited. We examined the functional and molecular adaptations to short- and long-term ET in elastic and muscular conduit arteries of db/db mice in relation to changes in cardiovascular risk factors. Diabetic mice and their controls were exercised at moderate intensity for 4 or 8weeks. The vasodilatory and contractile responses of thoracic aortae and femoral arteries isolated from the same animals were examined. Blood and aortic samples were used to measure hyperglycemia, oxidative stress, inflammation, dyslipidemia, protein expression of SOD isoforms, COX, eNOS, and Akt. Short-term ET improved nitric oxide (NO) mediated vasorelaxation in the aortae and femoral arteries of db/db mice in parallel with increased SOD2 and SOD3 expression, reduced oxidative stress and triglycerides, and independent of weight loss, glycemia, or inflammation. Long-term ET reduced body weight in parallel with reduced systemic inflammation and improved insulin sensitivity along with increased SOD1, Akt, and eNOS expression and improved NO vasorelaxation. Exercise did not restore NOS- and COX-independent vasodilatation in femoral arteries, nor did it mitigate the hypercontractility in the aortae of db/db mice; rather ET transiently increased contractility in association with upregulated COX-2. Long-term ET differentially affected the aortae and femoral arteries contractile responses. ET improved NO-mediated vasodilation in both arteries likely due to collective systemic effects. ET did not mitigate all diabetes-induced vasculopathies. Optimization of the ET regimen can help develop comprehensive management of type 2 diabetes.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

Multiple Sgip1 splice variants inhibit cannabinoid receptor 1 internalization

Alternative splicing can often result in the expression of distinct protein isoforms from a single gene, with specific composition and properties. SH3-containing GRB2-like protein 3-interacting protein 1 (Sgip1) is a brain-enriched protein that regulates clathrin-mediated endocytosis and interferes with the internalization of cannabinoid receptor 1. Several research groups have studied the physiological importance of Sgip1, and four Sgip1 protein isoforms have been described to date, while the NCBI Gene database predicts the expression of 20 splice variants from the Sgip1 gene in mice. In this work, we cloned 15 Sgip1 splice variants from the mouse brain, including 11 novel splice variants. The cloned splice variants differed in exon composition within two Sgip1 regions: the membrane phospholipid-binding domain and the proline-rich region. All the Sgip1 splice isoforms had similar stability and comparable ability to inhibit the internalization of cannabinoid receptor 1. None of the isoforms influenced the internalization of the µ-opioid receptor. We confirm the expression of Sgip1 splice variants described in previous studies or predicted in silico. Our data provide a basis for further studies exploring the significance of Sgip1 splicing, and we suggest a new classification of Sgip1 splice variants to unify their nomenclature.

Transcription Factor NFE2L1 Decreases in Glomerulonephropathies after Podocyte Damage.

Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.

Tissue-specific modulation of functional stress responsive proteins and organic osmolytes by a single or dual short-term high CO2 treatment during long-term cold storage Autumn Royal table grapes

Plant cell response to damage from environmental stressors that cause dehydration involves stress-related proteins and osmotically active compounds whose constitutive levels increase in association with the tolerance response mechanism. In table grape postharvest scope, the application of a second 3 d 20 kPa CO2 treatment significantly reduced the incidence and severity of long-term cold postharvest storage disorders in Autumn Royal cv. counteracting ultrastructural cell damage and restraining intracellular and membrane oxidative stress. In this sense, it may be of relevance to postharvest quality to know how this dual short-term high CO2 treatment modulates the cell stress responsive defense strategies, and whether these are tissue-dependent and timing-regulated. In this work, we studied the differential profile of protective osmolytes proline, glycine betaine, γ-aminobutyric acid and trehalose in the berry tissues. Likewise, we assessed the temporal and spatial expression and accumulation patterns of the thaumatin-like protein (VviTL1, VviOsmo) and dehydrin (VviDHN1a, VviDHN4, VviDHN2, VviDHN2D) isoforms in the whole berry tissues and rachis of cv. Autumn Royal table grapes during long-term cold storage period and evaluated the profile-shift by CO2 treatments. Their subcellular localization in the pericarp tissues was also determined by immunocytochemical transmission electron microscopy (TEM). The results showed that the cold-drought defense strategies activated by the short-term high CO2 are dose-dependent, tissue-specific and transcriptionally timing-regulated during long-term postharvest cold storage period in table grapes. The results also showed that the more short-term high CO2 treatment is applied to Autumn Royal grapes, the more microstructural evidence there is of enhanced tolerance to storage disorders in terms of thicker and denser cell wall that could contribute to restrain swelling, dehydration and osmotic stress.

Exploring the Expression and Prognosis of Mismatch Repair Proteins and PD-L1 in Colorectal Cancer in a Chinese Cohort.

Exploring the expression and prognosis of mismatch repair proteins and PD-L1 in colorectal cancer. A total of 272 patients with surgically resected CRC were enrolled in the study from January 2018 to May 2022 at Nanjing Drum Tower Hospital (The Affiliated Hospital of Nanjing University Medical School). Surgically resected samples were collected from patients along with general, clinicopathological, and imaging data for each patient. Immunohistochemistry (IHC) was used to detect expression of MSH2, MSH6, MLH1, and PMS2 proteins in tumor tissue. X-squared (X2) testing was performed to investigate the correlation between expression of MMR proteins and PD-L1 in CRC tumor tissues and clinicopathological characteristics. Correlation analysis was also used to compare the deletion of four MMR proteins in CRC tumor tissues. A survival curve and Log rank test were used to investigate the relationship between the expression of MMR proteins and PD-L1 with regard to CRC patient prognosis and survival. MMR protein expression deletion was correlated with tumor location, the degree of tissue differentiation, and TNM stage (P<0.05). PD-L1 expression was correlated with TNM stage (P<0.05). Correlation analysis of deletion of MMR protein isoform expression found that PMS2 deletion was significantly correlated with MLH1 deletion (P<0.05). Similarly, MSH2 deletion was significantly correlated with MSH6 deletion (P<0.05). PMS2 deletion was also found to be correlated with PD-L1 expression (P<0.05). Progression-free survival was found to be significantly longer in mismatch repair-proficient (pMMR) patients compared with mismatch repair-deficient (dMMR) patients. Deletion of MMR proteins and expression of PD-L1 are closely related to clinicopathological characteristics and overall prognosis of CRC patients. This suggests the relevance of MMR and PD-L1 as potential biomarkers for treatment of CRC patients.

Post-transcriptional regulation of erythropoiesis.

Erythropoiesis is a complex, precise, and lifelong process that is essential for maintaining normal body functions. Its strict regulation is necessary to prevent a variety of blood diseases. Normal erythropoiesis is precisely regulated by an intricate network that involves transcription levels, signal transduction, and various epigenetic modifications. In recent years, research on post-transcriptional levels in erythropoiesis has expanded significantly. The dynamic regulation of splicing transitions is responsible for changes in protein isoform expression that add new functions beneficial for erythropoiesis. RNA-binding proteins adapt the translation of transcripts to the protein requirements of the cell, yielding mRNA with dynamic translation efficiency. Noncoding RNAs, such as microRNAs and lncRNAs, are indispensable for changing the translational efficiency and/or stability of targeted mRNAs to maintain the normal expression of genes related to erythropoiesis. N6-methyladenosine-dependent regulation of mRNA translation plays an important role in maintaining the expression programs of erythroid-related genes and promoting erythroid lineage determination. This review aims to describe our current understanding of the role of post-transcriptional regulation in erythropoiesis and erythroid-associated diseases, and to shed light on the physiological and pathological implications of the post-transcriptional regulation machinery in erythropoiesis. These may help to further enrich our understanding of the regulatory network of erythropoiesis and provide new strategies for the diagnosis and treatment of erythroid-related diseases.

Synonymous alterations of cancer-associated Trp53 CpG mutational hotspots cause fatal developmental jaw malocclusions but no tumors in knock-in mice.

Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5-8 of Trp53, the mouse ortholog of human TP53, with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence; a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53-null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p < .0001). Since the latter phenotype also characterises murine Rett syndrome due to dysfunction of the trans-repressive MeCP2 methyl-CpG-binding protein, we hypothesise that CpG sites may exert non-coding phenotypic effects via pre-translational cis-interactions of 5-methylcytosine with methyl-binding proteins which regulate mRNA transcript initiation, expression or splicing, although direct effects on mRNA structure or translation are also possible.