A search for the in trans role of GraL, an Escherichia coli small RNA*

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL’s targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's β-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion’s activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.

Download Full-text

Functional genetic encoding of sulfotyrosine in mammalian cells

Nature Communications ◽

10.1038/s41467-020-18629-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xinyuan He ◽

Yan Chen ◽

Daisy Guiza Beltran ◽

Maia Kelly ◽

Bin Ma ◽

...

Keyword(s):

Mammalian Cells ◽

Biochemical Characterization ◽

Trna Synthetase ◽

Functional Verification ◽

Cellular Processes ◽

Genetic Encoding ◽

Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Download Full-text

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

10.1101/426809 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ana M. Oliveira Paiva ◽

Annemieke H. Friggen ◽

Liang Qin ◽

Roxanne Douwes ◽

Remus T. Dame ◽

...

Keyword(s):

Protein A ◽

Chromatin Protein ◽

Chromosomal Organization ◽

Cellular Processes ◽

Hu Proteins ◽

Architectural Proteins ◽

Chromatin Proteins

AbstractThe maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA, modulate its conformation and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of C. difficile have been characterized to date.Here, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.The present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.

Download Full-text

Mitogen Kinase Kinase (MKK7) controls cytokine production in vitro and in vivo in mice

10.1101/2021.06.25.449967 ◽

2021 ◽

Author(s):

Amada D. Caliz ◽

Hyung-Jin Yoo ◽

Anastassiia Vertii ◽

Cathy Tournier ◽

Roger J. Davis ◽

...

Keyword(s):

Cytokine Production ◽

Cell Types ◽

Minor Role ◽

Cellular Processes ◽

Mitogen Activated Protein ◽

And Migration ◽

Jnk Activation

Mitogen kinase kinase 4 (MKK4) and Mitogen kinase kinase 7 (MKK7) are members of the MAP2K family which can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both, c-Jun N-terminal Kinase (JNK) and p38 MAPK, whereas MKK7 only activates JNK in response to different stimuli. The stimuli as well as cell type determine the choice of MAP2K member that mediates the response. In a variety of cell types, the MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK7 and MKK4 contributes to innate immune response in macrophages as well as during inflammation in vivo. To address this question and elucidate the role of MKK7 and MKK4 in macrophage and in vivo, we developed MKK7- and MKK4-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for LPS induced cytokine production and migration which appears to be a major contributor to the inflammatory response in vivo. Whereas MKK4 plays a significant but minor role in cytokine production in vivo.

Download Full-text

The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2017432118 ◽

2021 ◽

Vol 118 (4) ◽

pp. e2017432118

Author(s):

Yalong Wang ◽

Kaixin He ◽

Baifa Sheng ◽

Xuqiu Lei ◽

Wanyin Tao ◽

...

Keyword(s):

Rna Splicing ◽

Intestinal Inflammation ◽

Paneth Cell ◽

Enteric Bacteria ◽

Paneth Cells ◽

Antimicrobial Protein ◽

Rna Helicases

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

Download Full-text

Volumetric Properties of Four-Stranded DNA Structures

Biology ◽

10.3390/biology10080813 ◽

2021 ◽

Vol 10 (8) ◽

pp. 813

Author(s):

Tigran V. Chalikian ◽

Robert B. Macgregor

Keyword(s):

Regulation Of Transcription ◽

Solvent Interactions ◽

Dna Structures ◽

Cellular Processes ◽

Molecular Determinants ◽

Telomere Biology ◽

The Stability

Four-stranded non-canonical DNA structures including G-quadruplexes and i-motifs have been found in the genome and are thought to be involved in regulation of biological function. These structures have been implicated in telomere biology, genomic instability, and regulation of transcription and translation events. To gain an understanding of the molecular determinants underlying the biological role of four-stranded DNA structures, their biophysical properties have been extensively studied. The limited libraries on volume, expansibility, and compressibility accumulated to date have begun to provide insights into the molecular origins of helix-to-coil and helix-to-helix conformational transitions involving four-stranded DNA structures. In this article, we review the recent progress in volumetric investigations of G-quadruplexes and i-motifs, emphasizing how such data can be used to characterize intra-and intermolecular interactions, including solvation. We describe how volumetric data can be interpreted at the molecular level to yield a better understanding of the role that solute–solvent interactions play in modulating the stability and recognition events of nucleic acids. Taken together, volumetric studies facilitate unveiling the molecular determinants of biological events involving biopolymers, including G-quadruplexes and i-motifs, by providing one more piece to the thermodynamic puzzle describing the energetics of cellular processes in vitro and, by extension, in vivo.

Download Full-text

Functional Role of AKNA: A Scoping Review

Biomolecules ◽

10.3390/biom11111709 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1709

Author(s):

Abrahán Ramírez-González ◽

Joaquín Manzo-Merino ◽

Carla Olbia Contreras-Ochoa ◽

Margarita Bahena-Román ◽

José Manasés Aguilar-Villaseñor ◽

...

Keyword(s):

Immune System ◽

Scoping Review ◽

Functional Role ◽

Inflammatory Diseases ◽

Data Extraction ◽

Negative Regulator ◽

Cellular Processes

Human akna encodes an AT-hook transcription factor whose expression participates in various cellular processes. We conducted a scoping review on the literature regarding the functional role of AKNA according to the evidence found in human and in vivo and in vitro models, stringently following the “PRISMA-ScR” statement recommendations. Methods: We undertook an independent PubMed literature search using the following search terms, AKNA OR AKNA ADJ gene OR AKNA protein, human OR AKNA ADJ functions. Observational and experimental articles were considered. The selected studies were categorized using a pre-determined data extraction form. A narrative summary of the evidence was produced. Results: AKNA modulates the expression of CD40 and CD40L genes in immune system cells. It is a negative regulator of inflammatory processes as evidenced by knockout mouse models and observational studies for several autoimmune and inflammatory diseases. Furthermore, AKNA contributes to the de-regulation of the immune system in cancer, and it has been proposed as a susceptibility genetic factor and biomarker in CC, GC, and HNSCC. Finally, AKNA regulates neurogenesis by destabilizing the microtubules dynamics. Conclusion: Our results provide evidence for the role of AKNA in various cellular processes, including immune response, inflammation, development, cancer, autoimmunity, and neurogenesis.

Download Full-text

UNC-45A is preferentially expressed in epithelial cells and binds to and co-localizes with interphase MTs

10.1101/615187 ◽

2019 ◽

Author(s):

Juri Habicht ◽

Ashley Mooneyham ◽

Mihir Shetty ◽

Xiaonan Zhang ◽

Vijayalakshmi Shridhar ◽

...

Keyword(s):

Epithelial Cells ◽

Ovarian Cancer Cells ◽

Mitotic Spindles ◽

Cellular Processes ◽

Interphase Cells ◽

Associated Proteins ◽

Chemotherapeutic Treatment

AbstractUNC-45A is a ubiquitously expressed protein highly conserved throughout evolution. Most of what we currently know about UNC-45A pertains to its role as a regulator of the actomyosin system. However, emerging studies from both our and other laboratories support a role of UNC-45A outside of actomyosin regulation. This includes studies showing that UNC-45A: regulates gene transcription, co-localizes and biochemically co-fractionates with gamma tubulin and regulates centrosomal positioning, is found in the same subcellular fractions where MT-associated proteins are, and is a mitotic spindle-associated protein with MT destabilizing activity in absence of the actomyosin system.Here, we extended our previous findings and show that UNC45A is variably expressed across a spectrum of cell lines with the highest level being found in HeLa cells and in ovarian cancer cells inherently paclitaxel-resistant. Furthermore, we show that UNC-45A is preferentially expressed in epithelial cells, localizes to mitotic spindles in clinical tumor specimens of cancer and co-localizes and co-fractionates with MTs in interphase cells independent of actin or myosin.In sum, we report alteration of UNC45A localization in the setting of chemotherapeutic treatment of cells with paclitaxel, and localization of UNC45A to MTs both in vitro and in vivo. These findings will be important to ongoing and future studies in the field that further identify the important role of UNC45A in cancer and other cellular processes.

Download Full-text

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22179364 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9364

Author(s):

Amada D. Caliz ◽

Hyung-Jin Yoo ◽

Anastassiia Vertii ◽

Ana C. Dolan ◽

Cathy Tournier ◽

...

Keyword(s):

Cytokine Production ◽

Cell Types ◽

Minor Role ◽

Cellular Processes ◽

Mitogen Activated Protein ◽

And Migration ◽

Jnk Activation

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

Download Full-text

Ras Suppressor-1 (RSU1) in Cancer Cell Metastasis: A Tale of a Tumor Suppressor

International Journal of Molecular Sciences ◽

10.3390/ijms21114076 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4076

Author(s):

Maria Louca ◽

Triantafyllos Stylianopoulos ◽

Vasiliki Gkretsi

Keyword(s):

Tumor Suppressor ◽

Cancer Cell ◽

Tumor Suppressor Genes ◽

Ras Oncogene ◽

Cellular Processes ◽

Cell Metastasis ◽

Cancer Types

Cancer is a multifactorial disease responsible for millions of deaths worldwide. It has a strong genetic background, as mutations in oncogenes or tumor suppressor genes contribute to the initiation of cancer development. Integrin signaling as well as the signaling pathway of Ras oncogene, have been long implicated both in carcinogenesis and disease progression. Moreover, they have been involved in the promotion of metastasis, which accounts for the majority of cancer-related deaths. Ras Suppressor-1 (RSU1) was identified as a suppressor of Ras-induced transformation and was shown to localize to cell-extracellular matrix adhesions. Recent findings indicate that its expression is elevated in various cancer types, while its role in regulating metastasis-related cellular processes remains largely unknown. Interestingly, there is no in vivo work in the field to date, and thus, all relevant knowledge stems from in vitro studies. In this review, we summarize recent studies using breast, liver and brain cancer cell lines and highlight the role of RSU1 in regulating cancer cell invasion.

Download Full-text

Protein synthesis regulation by leucine

Brazilian Journal of Pharmaceutical Sciences ◽

10.1590/s1984-82502010000100004 ◽

2010 ◽

Vol 46 (1) ◽

pp. 29-36 ◽

Cited By ~ 6

Author(s):

Daiana Vianna ◽

Gabriela Fullin Resende Teodoro ◽

Francisco Leonardo Torres-Leal ◽

Julio Tirapegui

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Mrna Translation ◽

Protein Translation ◽

Cellular Effects ◽

Cellular Processes ◽

High Protein Diets

In vivo and in vitro studies have demonstrated that high protein diets affect both protein synthesis and regulation of several cellular processes. The role of amino acids as substrate for protein synthesis has been established in the literature. However, the mechanism by which these amino acids modulate transcription and regulate the mRNA translation via mTOR-dependent signaling pathway has yet to be fully determined. It has been verified that mTOR is a protein responsible for activating a cascade of biochemical intracellular events which result in the activation of the protein translation process. Of the aminoacids, leucine is the most effective in stimulating protein synthesis and reducing proteolysis. Therefore, it promotes a positive nitrogen balance, possibly by favoring the activation of this protein. This amino acid also directly and indirectly stimulates the synthesis and secretion of insulin, enhancing its anabolic cellular effects. Therefore, this review aimed to identify the role of leucine in protein synthesis modulation and to discuss the metabolic aspects related to this aminoacid.

Download Full-text