scholarly journals Impairing activation of phospholipid synthesis by c-Fos interferes with glioblastoma cell proliferation

2020 ◽  
Vol 477 (23) ◽  
pp. 4675-4688
Author(s):  
César G. Prucca ◽  
Ana C. Racca ◽  
Fabiola N. Velazquez ◽  
Andrés M. Cardozo Gizzi ◽  
Lucia Rodríguez Berdini ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Glioblastoma Multiforme ◽  
Dominant Negative ◽  
Phospholipid Synthesis ◽  
Terminal Portion ◽  
Over Expression ◽  
Amino Terminal

Glioblastoma multiforme is the most aggressive type of tumor of the CNS with an overall survival rate of approximately one year. Since this rate has not changed significantly over the last 20 years, the development of new therapeutic strategies for the treatment of these tumors is peremptory. The over-expression of the proto-oncogene c-Fos has been observed in several CNS tumors including glioblastoma multiforme and is usually associated with a poor prognosis. Besides its genomic activity as an AP-1 transcription factor, this protein can also activate phospholipid synthesis by a direct interaction with key enzymes of their metabolic pathways. Given that the amino-terminal portion of c-Fos (c-Fos-NA: amino acids 1–138) associates to but does not activate phospholipid synthesizing enzymes, we evaluated if c-Fos-NA or some shorter derivatives are capable of acting as dominant-negative peptides of the activating capacity of c-Fos. The over-expression or the exogenous administration of c-Fos-NA to cultured T98G cells hampers the interaction between c-Fos and PI4K2A, an enzyme activated by c-Fos. Moreover, it was observed a decrease in tumor cell proliferation rates in vitro and a reduction in tumor growth in vivo when a U87-MG-generated xenograft on nude mice is intratumorally treated with recombinant c-Fos-NA. Importantly, a smaller peptide of 92 amino acids derived from c-Fos-NA retains the capacity to interfere with tumor proliferation in vitro and in vivo. Taken together, these results support the use of the N-terminal portion of c-Fos, or shorter derivatives as a novel therapeutic strategy for the treatment of glioblastoma multiforme.

scholarly journals Evaluation and detection of Brassicaceae leafy whey phytohormones efficacy for exploiting as a medium obtaining optimistic culture of fungi in vitro

2019 ◽  
Author(s):  
Rajesh Khanduji Jadhav
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Seed Germination ◽  
Secondary Metabolites ◽  
Nitrate Medium ◽  
Growth Optimization ◽  
Growth Of Fungi

The  DPJ  (Deproteinised  Juice)  or  whey  constituents    were  responsible  for  the  induction  of  growth  optimization  of  plants,  various  fungi  including  yeast,  Rhizobium  reported  by  earlier  workers. In  previous  experiments,  DPJ  maximised  the  growth  of  plants  and  seed  germination. During  present  investigation  the  carbohydrates,  amino  acids  and  protein   tests  were  taken  into  the  consideration. All the tests found positive.  Despite,  the  extract is  deproteinised,  still  there  was  persistence  of  few  proteins  and  amino  acids. The  collection  of  mycelia  grown  on  DPJ  was  filtered  and    the  culture  filtrates    recommended  to   use  in vitro  for  the  industrial  purpose  for   biomass  and  secondary  metabolites.   Experimental  DPJ  is  compared  with  the  glucose  nitrate  medium  as  control.  These  positive  tests  revealed  the suitability  of  DPJ  to  be  used  as  the  medium  for  the  growth  of  fungi.  Positive  amino  acid  tests  conspicuously  revealed  presence  of  phytohormones  in  members  of   Brassicaceae  DPJ  and  hence  advisable  to  be  utilised  for  the   plant  growth  in  vivo,  plant  callus  growth   and  cell  proliferation  of  mycelia  in  vitro. 

scholarly journals Hypoxia inhibits expression and function of mitochondrial thioredoxin 2 to promote pulmonary hypertension

2017 ◽  
Vol 312 (5) ◽  
pp. L599-L608 ◽  
Author(s):  
Sherry E. Adesina ◽  
Brandy E. Wade ◽  
Kaiser M. Bijli ◽  
Bum-Yong Kang ◽  
Clintoria R. Williams ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pulmonary Hypertension ◽  
Right Ventricular ◽  
Intracellular Signaling ◽  
Dominant Negative ◽  
Therapeutic Effects ◽  
Thioredoxin 2

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 h. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 wk. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.

Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2461-2468 ◽  
Author(s):  
Sandra E. Pike ◽  
Lei Yao ◽  
Joyce Setsuda ◽  
Karen D. Jones ◽  
Barry Cherney ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Endothelial Cell ◽  
Tumor Growth ◽  
Angiogenesis Inhibitors ◽  
Full Length ◽  
Endothelial Cell Proliferation ◽  
Proliferation In Vitro

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

scholarly journals Down-Regulating HAUS6 Suppresses Cell Proliferation by Activating the p53/p21 Pathway in Colorectal Cancer

2022 ◽  
Vol 9 ◽  
Author(s):  
Aling Shen ◽  
Liya Liu ◽  
Yue Huang ◽  
Zhiqing Shen ◽  
Meizhu Wu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Over Expression ◽  
Mrna And Protein Expression

Background: HAUS6 participates in microtubule-dependent microtubule amplification, but its role in malignancies including colorectal cancer (CRC) has not been explored. We therefore assessed the potential oncogenic activities of HAUS6 in CRC.Results: HAUS6 mRNA and protein expression is higher in CRC tissues, and high HAUS6 expression is correlated with shorter overall survival in CRC patients. HAUS6 knockdown in CRC cell lines suppressed cell growth in vitro and in vivo by inhibiting cell viability, survival and arresting cell cycle progression at G0/G1, while HAUS6 over-expression increased cell viability. We showed that these effects are dependent on activation of the p53/p21 signalling pathway by reducing p53 and p21 degradation. Moreover, combination of HAUS6 knockdown and 5-FU treatment further enhanced the suppression of cell proliferation of CRC cells by increasing activation of the p53/p21 pathway.Conclusion: Our study highlights a potential oncogenic role for HAUS6 in CRC. Targeting HAUS6 may be a promising novel prognostic marker and chemotherapeutic target for treating CRC patients.

scholarly journals The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

1993 ◽  
Vol 13 (12) ◽  
pp. 7874-7880 ◽  
Author(s):  
S Pesce ◽  
R Benezra
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Dominant Negative Activity

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

scholarly journals The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 934-940 ◽  
Author(s):  
Wilson B. Muse ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Nitrogen Assimilation ◽  
Factor Xa ◽  
Klebsiella Aerogenes ◽  
Reading Frame ◽  
Amino Terminal ◽  
Control Protein

ABSTRACT The nitrogen assimilation control protein (NAC) fromKlebsiella aerogenes or Escherichia coli(NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within thenac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activatehut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACKfragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

Evaluation and detection of Brassicaceae leafy whey phytohormones efficacy for exploiting as a medium obtaining optimistic culture of fungi in vitro

2019 ◽  
pp. 1-5
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Seed Germination ◽  
Secondary Metabolites ◽  
Nitrate Medium ◽  
Growth Optimization ◽  
Growth Of Fungi

The DPJ (Deproteinised Juice) or whey constituents were responsible for the induction of growth optimization of plants, various fungi including yeast, Rhizobium reported by earlier workers. In previous experiments, DPJ maximised the growth of plants and seed germination. During present investigation the carbohydrates, amino acids and protein tests were taken into the consideration. All the tests found positive. Despite, the extract is deproteinised, still there was persistence of few proteins and amino acids. The collection of mycelia grown on DPJ was filtered and the culture filtrates recommended to use in vitro for the industrial purpose for biomass and secondary metabolites. Experimental DPJ is compared with the glucose nitrate medium as control. These positive tests revealed the suitability of DPJ to be used as the medium for the growth of fungi. Positive amino acid tests conspicuously revealed presence of phytohormones in members of Brassicaceae DPJ and hence advisable to be utilised for the plant growth in vivo, plant callus growth and cell proliferation of mycelia in vitro.

scholarly journals Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369 ◽  
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

scholarly journals Engineering posttranslational proofreading to discriminate nonstandard amino acids

2018 ◽  
Vol 115 (3) ◽  
pp. 619-624 ◽  
Author(s):  
Aditya M. Kunjapur ◽  
Devon A. Stork ◽  
Erkin Kuru ◽  
Oscar Vargas-Rodriguez ◽  
Matthieu Landon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Trna Synthetase ◽  
Genetically Engineered ◽  
Major Advance ◽  
Biological Processes ◽  
Trna Synthetases ◽  
Rational Engineering ◽  
Amino Terminal

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called “posttranslational proofreading” to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.

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