Live-cell protein engineering with an ultra-short split intein.

Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein trans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.

PLA2G12A as a Novel Biomarker for Colorectal Cancer with Prognostic Relevance.

Metastasis is the leading cause of colorectal cancer (CRC)-related deaths. Therefore, the identification of accurate biomarkers predictive of metastasis is needed to better stratify high-risk patients to provide preferred management and reduce mortality. In this study, we identified 13 new genes that modified circulating tumor cell numbers using a genome-wide genetic screen in a whole animal CRC model. Candidate genes were subsequently evaluated at the gene expression level in both an internal human CRC cohort of 153 patients and an independent cohort from the TCGA including 592 patients. Interestingly, the expression of one candidate, PLA2G12A, significantly correlated with both the time to recurrence and overall survival in our CRC cohort, with its low expression being an indicator of a poor clinical outcome. By examining the TCGA cohort, we also found that low expression of PLA2G12A was significantly enriched in epithelial-mesenchymal transition signatures. Finally, the candidate functionality was validated in vitro using three different colon cancer cell lines, revealing that PLA2G12A deficiency increases cell proliferation, migration, and invasion. Overall, our study identifies PLA2G12A as a prognostic biomarker of early-stage CRC, providing evidence that its deficiency promotes tumor growth and dissemination.

MicroRNAs Present in Malignant Pleural Fluid Increase the Migration of Normal Mesothelial Cells In Vitro and May Help Discriminate between Benign and Malignant Effusions.

The sensitivity of pleural fluid (PF) analyses for the diagnosis of malignant pleural effusions (MPEs) is low to moderate. Knowledge about the pathobiology and molecular characteristics of this condition is limited. In this study, the crosstalk between stromal cells and tumor cells was investigated in vitro in order to reveal factors that are present in PF which can mediate MPE formation and aid in discriminating between benign and malignant etiologies. Eighteen PF samples, in different proportions, were exposed in vitro to mesothelial MeT-5A cells to determine the biological effects on these cells. Treatment of normal mesothelial MeT-5A cells with malignant PF increased cell viability, proliferation, and migration, and activated different survival-related signaling pathways. We identified differentially expressed miRNAs in PF samples that could be responsible for these changes. Consistently, bioinformatics analysis revealed an enrichment of the discovered miRNAs in migration-related processes. Notably, the abundance of three miRNAs (miR-141-3p, miR-203a-3, and miR-200c-3p) correctly classified MPEs with false-negative cytological examination results, indicating the potential of these molecules for improving diagnosis. Malignant PF produces phenotypic and functional changes in normal mesothelial cells. These changes are partly mediated by certain miRNAs, which, in turn, could serve to differentiate malignant from benign effusions.

Cdc48/p97 segregase is modulated by cyclin-dependent kinase to determine cyclin fate during G1 progression.

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short-lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1-cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk-target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1-cyclin levels and activity. Cdk-dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear.

A Whi7-anchored loop controls the G1 Cdk-cyclin complex at start.

Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry.

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast.

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.

Applications of CRISPR Technology to Breast Cancer and Triple Negative Breast Cancer Research.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has transformed oncology research in many ways. Breast cancer is the most prevalent malignancy globally and triple negative breast cancer (TNBC) is one of the most aggressive subtypes with numerous challenges still to be faced. In this work, we have explained what CRISPR consists of and listed its applications in breast cancer while focusing on TNBC research. These are disease modelling, the search for novel genes involved in tumour progression, sensitivity to drugs and immunotherapy response, tumour fitness, diagnosis, and treatment. Additionally, we have listed the current delivery methods employed for the delivery of CRISPR systems in vivo. Lastly, we have highlighted the limitations that CRISPR technology is subject to and the future directions that we envisage. Overall, we have provided a round summary of the aspects concerning CRISPR in breast cancer/TNBC research.

Glutamatergic signaling maintains the epithelial phenotype of proximal tubular cells.

Epithelial-mesenchymal transition (EMT) contributes to the progression of renal tubulointerstitial fibrosis. The N-methyl-d-aspartate receptor (NMDAR), which is present in proximal tubular epithelium, is a glutamate receptor that acts as a calcium channel. Activation of NMDAR induces actin rearrangement in cells of the central nervous system, but whether it helps maintain the epithelial phenotype of the proximal tubule is unknown. Here, knockdown of NMDAR1 in a proximal tubule cell line (HK-2) induced changes in cell morphology, reduced E-cadherin expression, and increased α-SMA expression. Induction of EMT with TGF-ß1 led to downregulation of both E-cadherin and membrane-associated ß-catenin, reorganization of F-actin, expression of mesenchymal markers de novo, upregulation of Snail1, and increased cell migration; co-treatment with NMDA attenuated all of these changes. Furthermore, NMDA reduced TGF-ß1-induced phosphorylation of Erk1/2 and Akt and the activation of Ras, suggesting that NMDA antagonizes TGF-ß1-induced EMT by inhibiting the Ras-MEK pathway. In the unilateral ureteral obstruction model, treatment with NMDA blunted obstruction-induced upregulation of α-SMA, FSP1, and collagen I and downregulation of E-cadherin. Taken together, these results suggest that NMDAR plays a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.

RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure.

DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the increasing resolution offered by super-resolution microscopy, the optimal maintenance of chromatin structure within the nucleus is essential for accuracy in measurements and interpretation of data. However, standard 3D-FISH requires potentially destructive heat denaturation in the presence of chaotropic agents such as formamide to allow access to the DNA strands for labeled FISH probes. To avoid the need to heat-denature, we developed Resolution After Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease digestion to generate single-stranded target DNA for efficient probe binding over a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of nuclear proteins or the detection of RNAs. Here, we provide detailed procedures for RASER-FISH in mammalian cultured cells to detect single loci, chromatin tracks and topologically associating domains with conventional and super-resolution 3D structured illumination microscopy. Moreover, we provide a validation and characterization of our method, demonstrating excellent preservation of chromatin structure and nuclear integrity, together with improved hybridization efficiency, compared with classic 3D-FISH protocols.

Are Transcription Factors Plausible Oncotargets for Triple Negative Breast Cancers?

Breast cancer (BC) is the most diagnosed cancer worldwide and one of the main causes of cancer deaths. BC is a heterogeneous disease composed of different BC intrinsic subtypes such as triple-negative BC (TNBC), which is one of the most aggressive subtypes and which lacks a targeted therapy. Recent comprehensive analyses across cell types and cancer types have outlined a vast network of protein-protein associations between transcription factors (TFs). Not surprisingly, protein-protein networks central to oncogenesis and disease progression are highly altered during TNBC pathogenesis and are responsible for the activation of oncogenic programs, such as uncontrollable proliferation, epithelial-to-mesenchymal transition (EMT) and stemness. From the therapeutic viewpoint, inhibiting the interactions between TFs represents a very significant challenge, as the contact surfaces of TFs are relatively large and featureless. However, promising tools have emerged to offer a solution to the targeting problem. At the clinical level, some TF possess diagnostic and prognostic value in TNBC. In this review, we outline the recent advances in TFs relevant to TNBC growth and progression. Moreover, we highlight different targeting approaches to inhibit these TFs. Furthermore, the validity of such TFs as clinical biomarkers has been explored. Finally, we discuss how research is likely to evolve in the field.

RANKL increases vascular smooth muscle cell calcification through a RANK-BMP4-dependent pathway.

Vascular calcification commonly associated with several pathologies and it has been suggested to be similar to bone mineralization. The axis RANKL-OPG (receptor activator of nuclear factor kappaB ligand-osteoprotegerin) finely controls bone turnover. RANKL has been suggested to increase vascular calcification, but direct evidence is missing. Thus, in the present work, we assess the effect of RANKL in vascular smooth muscle cell (VSMC) calcification. VSMCs incubated with RANKL showed a dose-dependent increase in calcification, which was abolished by coincubation with OPG. To test whether the effect was mediated by signaling to its receptor, knockdown of RANK was accomplished by short hairpin (sh)RNA. Indeed, cells lacking RANK showed no increases in vascular calcification when incubated with RANKL. To further elucidate the mechanism by which RANK activation increases calcification, we blocked both nuclear factor (NF)-kappaB activation pathways. Only IKKalpha inactivation inhibited calcification, pointing to an involvement of the alternative NF-kappaB activation pathway. Furthermore, RANKL addition increased bone morphogenetic protein (BMP)4 expression in VSMCs, and that increase disappeared in cells lacking RANK or IKKalpha. The increase in calcification was also blunted by Noggin, pointing to a mediation of BMP4 in the calcification induced by RANKL. Furthermore, in an in vivo model, the increase in vascular calcium content was parallel to an increase in RANKL and BMP4 expression, which was localized in calcified areas. However, blood levels of the ratio RANKL/OPG did not change. We conclude that RANKL increases vascular smooth muscle cell calcification by binding to RANK and increasing BMP4 production through activation of the alternative NF-kappaB pathway.

An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 (Nrf1) Altering the DNA Methylation State and Correct Embryonic Development.

We have established a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify novel genes playing a role in epigenetic regulation in mammals. We hypothesize that the ENU mutagenesis screen will lead to the discovery of unknown genes responsible of the maintenance of the epigenetic state as the genes found are modifiers of variegation of the transgene green fluorescent protein (GFP) expression in erythrocytes, which are named MommeD. Here we report the generation of a novel mutant mouse line, MommeD46, that carries a new missense mutation producing an amino acid transversion (L71P) in the dimerization domain of Nuclear Respiratory Factor 1 (Nrf1). The molecular characterization of the mutation reveals a decrease in the Nrf1 mRNA levels and a novel role of Nrf1 in the maintenance of the DNA hypomethylation in vivo. The heritability of the mutation is consistent with paternal imprinting and haploinsufficiency. Homozygous mutants display embryonic lethality at 14.5 days post-coitum and developmental delay. This work adds a new epi-regulatory role to Nrf1 and uncovers unknown phenotypical defects of the Nrf1 hypomorph. The generated mouse line represents a valuable resource for studying NRF1-related diseases.

Microenvironmental Reactive Oxygen Species in Colorectal Cancer: Involved Processes and Therapeutic Opportunities.

Colorectal cancer (CRC) is the fourth most common cause of cancer deaths worldwide. Although screening programs have reduced mortality rates, there is a need for research focused on finding the main factors that lead primary CRC to progress and metastasize. During tumor progression, malignant cells modify their habitat, corrupting or transforming cells of different origins and creating the tumor microenvironment (TME). Cells forming the TME like macrophages, neutrophils, and fibroblasts generate reactive oxygen species (ROS) that modify the cancer niche. The effects of ROS in cancer are very diverse: they promote cellular proliferation, epithelial-to-mesenchymal transition (EMT), evasion of cell death programs, migration, and angiogenesis. Due to the multifaceted role of ROS in cancer cell survival and function, ROS-modulating agents such as antioxidants or pro-oxidants could have therapeutic potential in cancer prevention and/or as a complement to systemic treatments. In this review, we will examine the main ROS producer cells and their effects on cancer progression and metastasis. Furthermore, we will enumerate the latest clinical trials where pro-oxidants and antioxidants have therapeutic uses in CRC.

Diving into the Pleural Fluid: Liquid Biopsy for Metastatic Malignant Pleural Effusions.

Liquid biopsy is emerging as a promising non-invasive diagnostic tool for malignant pleural effusions (MPE) due to the low sensitivity of conventional pleural fluid (PF) cytological examination and the difficulty to obtain tissue biopsies, which are invasive and require procedural skills. Currently, liquid biopsy is increasingly being used for the detection of driver mutations in circulating tumor DNA (ctDNA) from plasma specimens to guide therapeutic interventions. Notably, malignant PF are richer than plasma in tumor-derived products with potential clinical usefulness, such as ctDNA, micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs), and circulating tumor cells (CTC). Tumor-educated cell types, such as platelets and macrophages, have also been added to this diagnostic armamentarium. Herein, we will present an overview of the role of the preceding biomarkers, collectively known as liquid biopsy, in PF samples, as well as the main technical approaches used for their detection and quantitation, including a proper sample processing. Technical limitations of current platforms and future perspectives in the field will also be addressed. Using PF as liquid biopsy shows promise for use in current practice to facilitate the diagnosis and management of metastatic MPE.

Stress granules display bistable dynamics modulated by Cdk.

Stress granules (SGs) are conserved biomolecular condensates that originate in response to many stress conditions. These membraneless organelles contain nontranslating mRNAs and a diverse subproteome, but our knowledge of their regulation and functional relevance is still incipient. Here, we describe a mutual-inhibition interplay between SGs and Cdc28, the budding yeast Cdk. Among Cdc28 interactors acting as negative modulators of Start, we have identified Whi8, an RNA-binding protein that localizes to SGs and recruits the mRNA of CLN3, the most upstream G1 cyclin, for efficient translation inhibition and Cdk inactivation under stress. However, Whi8 also contributes to recruiting Cdc28 to SGs, where it acts to promote their dissolution. As predicted by a mutual-inhibition framework, the SG constitutes a bistable system that is modulated by Cdk. Since mammalian cells display a homologous mechanism, we propose that the opposing functions of specific mRNA-binding proteins and Cdk's subjugate SG dynamics to a conserved hysteretic switch.

Sustained activation of renal N-methyl-D-aspartate receptors decreases vitamin D synthesis: a possible role for glutamate on the onset of secondary HPT.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are tetrameric amino acid receptors that act as membrane calcium channels. The presence of the receptor has been detected in the principal organs responsible for calcium homeostasis (kidney, bone, and parathyroid gland), pointing to a possible role in mineral metabolism. The aim of this study was to test the effect of NMDAR activation in the kidney and on 1,25(OH)2D3 synthesis. We determined the presence of NMDAR subunits in HK-2 (human kidney cells) cells and proved its functionality. NMDA treatment for 4 days induced a decrease in 1α-hydroxylase levels and 1,25(OH)2D3 synthesis through the activation of the MAPK/ERK pathway in HK-2 cells. In vivo administration of NMDA for 4 days also caused a decrease in blood 1,25(OH)2D3 levels in healthy animals and an increase in blood PTH levels. This increase in PTH induced a decrease in the urinary excretion of calcium and an increase in urinary excretion of phosphorous and sodium as well as in diuresis. Bone turnover markers also increased. Animals with 5/6 nephrectomy showed low levels of renal 1α-hydroxylase as well as high levels of renal glutamate compared with healthy animals. In conclusion, NMDAR activation in the kidney causes a decrease in 1,25(OH)2D3 synthesis, which induces an increase on PTH synthesis and release. In animals with chronic kidney disease, high renal levels of glutamate could be involved in the downregulation of 1α-hydroxylase expression.

Determinants of Sensitivity to Radiotherapy in Endometrial Cancer.

Radiotherapy is one of the cornerstone treatments for endometrial cancer and has successfully diminished the risk of local recurrences after surgery. However, a considerable percentage of patients suffers tumor relapse due to radioresistance mechanisms. Knowledge about the molecular determinants that confer radioresistance or radiosensitivity in endometrial cancer is still partial, as opposed to other cancers. In this review, we have highlighted different central cellular signaling pathways and processes that are known to modulate response to radiotherapy in endometrial cancer such as PI3K/AKT, MAPK and NF-κB pathways, growth factor receptor signaling, DNA damage repair mechanisms and the immune system. Moreover, we have listed different clinical trials employing targeted therapies against some of the aforementioned signaling pathways and members with radiotherapy. Finally, we have identified the latest advances in radiotherapy that have started being utilized in endometrial cancer, which include modern radiotherapy and radiogenomics. New molecular and genetic studies in association with the analysis of radiation responses in endometrial cancer will assist clinicians in taking suitable decisions for each individual patient and pave the path for personalized radiotherapy.

Prognostic Factors Involved in the Epithelial-Mesenchymal Transition Process in Colorectal Cancer Have a Preponderant Role in Oxidative Stress: A Systematic Review and Meta-Analysis.

Epithelial-to-mesenchymal transition (EMT) is one of the most accepted mechanisms leading to metastasis, which is responsible for most of the cancer-related deaths. In order to identify EMT-related biomarkers able to predict clinical outcomes in colorectal cancer (CRC), a systematic review and meta-analysis of prognostic factors associated to overall survival (OS) and progression free survival (PFS) was conducted. The systematic literature search included studies from June 2014 to June 2019 available at PubMed and Scopus databases. Meta-analysis was performed for those markers appearing in minimum three works with a total number of 8656 participants. The rest were enlisted and subjected to functional enrichment. We identified nine clinical biomarkers and 73 EMT-related molecular biomarkers associated to OS and/or PFS in CRC. The significant enrichment of biomarkers found involved in cellular oxidoreductase activity suggests that ROS generation plays an active role in the EMT process. Clinical practice needs new biomarkers with a reliable prognostic value able to predict clinical outcomes in CRC. Our integrative work supports the role of oxidative stress in tumorigenesis and EMT progress highlighting the importance of deciphering this specific mechanism to get a better understanding of metastasis.

Competition in the chaperone-client network subordinates cell-cycle entry to growth and stress.

The precise coordination of growth and proliferation has a universal prevalence in cell homeostasis. As a prominent property, cell size is modulated by the coordination between these processes in bacterial, yeast, and mammalian cells, but the underlying molecular mechanisms are largely unknown. Here, we show that multifunctional chaperone systems play a concerted and limiting role in cell-cycle entry, specifically driving nuclear accumulation of the G1 Cdk-cyclin complex. Based on these findings, we establish and test a molecular competition model that recapitulates cell-cycle-entry dependence on growth rate. As key predictions at a single-cell level, we show that availability of the Ydj1 chaperone and nuclear accumulation of the G1 cyclin Cln3 are inversely dependent on growth rate and readily respond to changes in protein synthesis and stress conditions that alter protein folding requirements. Thus, chaperone workload would subordinate Start to the biosynthetic machinery and dynamically adjust proliferation to the growth potential of the cell.