Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability.

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes.

Recurrence of high-grade prostate cancer after radiotherapy is a significant clinical problem, resulting in increased morbidity and reduced patient survival. The molecular mechanisms of radiation resistance are being elucidated through the study of microRNA (miR) that negatively regulate gene expression. We performed bioinformatics analyses of The Cancer Genome Atlas (TCGA) dataset to evaluate the association between miR-106a and its putative target lipopolysaccharide-induced TNF-α factor (LITAF) in prostate cancer. We characterized the function of miR-106a through in vitro and in vivo experiments and employed transcriptomic analysis, western blotting, and 3'UTR luciferase assays to establish LITAF as a bona fide target of miR-106a. Using our well-characterized radiation-resistant cell lines, we identified that miR-106a was overexpressed in radiation-resistant cells compared to parental cells. In the TCGA, miR-106a was significantly elevated in high-grade human prostate tumors relative to intermediate- and low-grade specimens. An inverse correlation was seen with its target, LITAF. Furthermore, high miR-106a and low LITAF expression predict for biochemical recurrence at 5 years after radical prostatectomy. miR-106a overexpression conferred radioresistance by increasing proliferation and reducing senescence, and this was phenocopied by knockdown of LITAF. For the first time, we describe a role for miRNA in upregulating ATM expression. LITAF, not previously attributed to radiation response, mediates this interaction. This route of cancer radioresistance can be overcome using the specific ATM kinase inhibitor, KU-55933. Our research provides the first report of miR-106a and LITAF in prostate cancer radiation resistance and high-grade disease, and presents a viable therapeutic strategy that may ultimately improve patient outcomes.

15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory/anti-neoplastic prostaglandin that functions through covalent binding to cysteine residues of various target proteins. We previously showed that 15d-PGJ2 mediated anti-inflammatory responses are dependent on the translational inhibition through its interaction with eIF4A (Kim et al., 2007). Binding of 15d-PGJ2 to eIF4A specifically blocks the interaction between eIF4G and eIF4A, which leads to the formation of stress granules (SGs), which then cluster mRNAs with inhibited translation. Here, we show that the binding between 15d-PGJ2 and eIF4A specifically blocks the interaction between the MIF4G domain of eIF4G and eIF4A. To reveal the mechanism of this interaction, we used computational simulation-based docking studies and identified that the carboxyl tail of 15d-PGJ2 could stabilize the binding of 15d-PGJ2 to eIF4A through arginine 295 of eIF4A, which is the first suggestion that the 15d-PGJ2 tail plays a physiological role. Interestingly, the putative 15d-PGJ2 binding site on eiF4A is conserved across many species, suggesting a biological role. Our data propose that studying 15d-PGJ2 and its targets may uncover new therapeutic approaches in anti-inflammatory drug discovery.

We present an integrated approach that predicts and validates novel anti-cancer drug targets. We first built a classifier that integrates a variety of genomic and systematic datasets to prioritize drug targets specific for breast, pancreatic and ovarian cancer. We then devised strategies to inhibit these anti-cancer drug targets and selected a set of targets that are amenable to inhibition by small molecules, antibodies and synthetic peptides. We validated the predicted drug targets by showing strong anti-proliferative effects of both synthetic peptide and small molecule inhibitors against our predicted targets.

Characterizing the spatial organization of the human mitochondrial proteome will enhance our understanding of mitochondrial functions at the molecular level and provide key insight into protein-disease associations. However, the sub-organellar location and possible association with mitochondrial diseases are not annotated for most mitochondrial proteins. Here, we characterized the functional and spatial organization of mitochondrial proteins by assessing their position in the Mitochondrial Protein Functional (MPF) network. Network position was assigned to the MPF network and facilitated the determination of sub-organellar location and functional organization of mitochondrial proteins. Moreover, network position successfully identified candidate disease genes of several mitochondrial disorders. Thus, our data support the use of network position as a novel method to explore the molecular function and pathogenesis of mitochondrial proteins.

In December 2019, the COVID-19 outbreak started in China and rapidly spread around the world. Many studies have been conducted to understand the clinical characteristics of COVID-19, and recently postinfection sequelae of this disease have begun to be investigated. However, there is little consensus on the longitudinal changes of lasting physical or psychological symptoms from prior COVID-19 infection.

Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers.

MicroRNAs are small non‑coding RNA molecules which act as modulators of gene function, and have been identified as playing important roles in cancer as both tumor suppressors and oncogenes. The present study aimed to examine the role of miR‑198 in prostate cancer aggression by analyzing how it influences several hallmarks of cancer. Abundance of miR‑198 in prostate cancer and association with clinical characteristics was analyzed using a CPC‑Gene prostate cancer dataset. Overexpression of miR‑198 was performed using transient transfection of miR‑198 mimic prior to assaying proliferation, cell cycle, and colony formation in LNCaP and DU145 cell lines using standard protocols. In vivo tumor formation in athymic nude mice was examined using LNCaP xenografts with stable overexpression conferred using lentiviral miR‑198 transduction. Protein and mRNA abundance of MIB1 was determined using western blotting and RT‑qPCR respectively, while miR‑198 binding to MIB1 was validated using a luciferase reporter assay. miR‑198 abundance was lower in high Gleason grade prostate cancer relative to intermediate and low‑grade cancer. Overexpression of miR‑198 diminished proliferation of prostate cancer cell lines, increased G0/G1 cell cycle arrest, and significantly impaired colony formation. Elevated miR‑198 abundance was also demonstrated to impair tumor formation in vivo using LNCaP xenografts. Mindbomb E3 ubiquitin protein ligase 1 (MIB1) was demonstrated to be directly targeted by miR‑198, and knockdown of MIB1 recapitulated the effects of miR‑198 on proliferation and colony formation. The present evidence supports miR‑198 as an important tumor suppressor in prostate cancer, and demonstrates for the first time that it acts by targeting MIB1. The present study reinforces the importance and complexity of miRNA in regulating prostate cancer aggression.

The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ(0)) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ(0) cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions.

Tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous target in cardiovascular diseases. Although it is involved in cardiovascular metabolism and mitochondrial biology, its mechanisms of action are unclear. We investigated how BH4 regulates cardiovascular metabolism using an unbiased multiple proteomics approach with a sepiapterin reductase knock-out (Spr-/-) mouse as a model of BH4 deficiency. Spr-/- mice exhibited a shortened life span, cardiac contractile dysfunction, and morphological changes. Multiple proteomics and systems-based data-integrative analyses showed that BH4 deficiency altered cardiac mitochondrial oxidative phosphorylation. Along with decreased transcription of major mitochondrial biogenesis regulatory genes, including Ppargc1a, Ppara, Esrra, and Tfam, Spr-/- mice exhibited lower mitochondrial mass and severe oxidative phosphorylation defects. Exogenous BH4 supplementation, but not nitric oxide supplementation or inhibition, rescued these cardiac and mitochondrial defects. BH4 supplementation also recovered mRNA and protein levels of PGC1α and its target proteins involved in mitochondrial biogenesis (mtTFA and ERRα), antioxidation (Prx3 and SOD2), and fatty acid utilization (CD36 and CPTI-M) in Spr-/- hearts. These results indicate that BH4-activated transcription of PGC1α regulates cardiac energy metabolism independently of nitric oxide and suggests that BH4 has therapeutic potential for cardiovascular diseases involving mitochondrial dysfunction.

Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide-binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreatic cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action, including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage-dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads, and it provides a rich data set covering phenotypes for the inhibition of thousands of interactions.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.

In December 2019, the COVID-19 outbreak started in China and rapidly spread around the world. Lack of a vaccine or optimized intervention raised the importance of characterizing risk factors and symptoms for the early identification and successful treatment of patients with COVID-19.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.