scholarly journals Regulation of antimycin biosynthesis is controlled by the ClpXP protease

2019 ◽  
Author(s):  
Bohdan Bilyk ◽  
Sora Kim ◽  
Asif Fazal ◽  
Tania A. Baker ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Rna Polymerase ◽  
Morphological Differentiation ◽  
Dynamic Responses ◽  
Biosynthetic Pathways ◽  
Recognition Sequence ◽  
Data Link ◽  
Σ Factor

AbstractThe survival of any microbe relies upon its ability to respond to environmental change. Use of Extra Cytoplasmic Function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors; nearly all of which regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstitute in vitro ClpXP proteolysis of σAntA, but not a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo is enhanced by removal of the ClpXP recognition sequence, and that levels of the protein rise when cellular ClpXP protease activity is abolished. These data establish direct proteolysis as an alternative and thus far unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.ImportanceNatural products produced by Streptomyces species underpin many industrially- and medically-important compounds. However, the majority of the ~30 biosynthetic pathways harboured by an average species are not expressed in the laboratory. This undiscovered biochemical diversity is believed to comprise an untapped resource for natural products drug discovery. A major roadblock preventing the exploitation of unexpressed biosynthetic pathways is a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

scholarly journals Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Bohdan Bilyk ◽  
Sora Kim ◽  
Asif Fazal ◽  
Tania A. Baker ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Rna Polymerase ◽  
Natural Product ◽  
Morphological Differentiation ◽  
Dynamic Responses ◽  
Biosynthetic Pathways ◽  
Recognition Sequence ◽  
Σ Factor

ABSTRACT The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntA in vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation. IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

scholarly journals Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1

2009 ◽  
Vol 191 (12) ◽  
pp. 3763-3771 ◽  
Author(s):  
Mohammed Dehbi ◽  
Gregory Moeck ◽  
Francis F. Arhin ◽  
Pascale Bauda ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Growth Inhibitory ◽  
Σ Factor ◽  
Transcription In Vitro

ABSTRACT The primary sigma factor of Staphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSA both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSA that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSA to DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

scholarly journals Properties of Bacillus subtilis σA Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

2004 ◽  
Vol 186 (8) ◽  
pp. 2366-2375 ◽  
Author(s):  
Hsin-Hsien Hsu ◽  
Wei-Cheng Huang ◽  
Jia-Perng Chen ◽  
Liang-Yin Huang ◽  
Chai-Fong Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Activity ◽  
Transcription Activity ◽  
N Terminus ◽  
Σ Factor

ABSTRACT σ factors in the σ70 family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis σA, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σA and greatly reduced the transcription activity of the truncated σA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σA in RNA polymerase.

scholarly journals The C Terminus of ς32 Is Not Essential for Degradation by FtsH

2001 ◽  
Vol 183 (20) ◽  
pp. 5911-5917 ◽  
Author(s):  
Toshifumi Tomoyasu ◽  
Florence Arsène ◽  
Teru Ogura ◽  
Bernd Bukau
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Recognition Sequence ◽  
Mutant Proteins ◽  
C Terminus ◽  
Polymerase Binding ◽  
Stress Dependent

ABSTRACT A key step in the regulation of heat shock genes inEscherichia coli is the stress-dependent degradation of the heat shock promoter-specific ς32 subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including ς32, as degradation signals for AAA proteases. We investigated the role of the C terminus of ς32 in FtsH-dependent degradation by analysis of C-terminally truncated ς32 mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of ς32 did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of ς32 was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated ς32 mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of ς32 in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.

Natural Anti-inflammatory compounds as Drug candidates in Alzheimer’s disease

2020 ◽  
Vol 27 ◽  
Author(s):  
Reyaz Hassan Mir ◽  
Abdul Jalil Shah ◽  
Roohi Mohi-ud-din ◽  
Faheem Hyder Potoo ◽  
Mohd. Akbar Dar ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Natural Products ◽  
Protective Action ◽  
New Drugs ◽  
Huperzine A ◽  
Brain Disorder ◽  
Anti Inflammatory

: Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder characterized by memory impairment, dementia, oxidative stress in elderly people. Currently, only a few drugs are available in the market with various adverse effects. So to develop new drugs with protective action against the disease, research is turning to the identification of plant products as a remedy. Natural compounds with anti-inflammatory activity could be good candidates for developing effective therapeutic strategies. Phytochemicals including Curcumin, Resveratrol, Quercetin, Huperzine-A, Rosmarinic acid, genistein, obovatol, and Oxyresvertarol were reported molecules for the treatment of AD. Several alkaloids such as galantamine, oridonin, glaucocalyxin B, tetrandrine, berberine, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in-vivo. In conclusion, natural products from plants represent interesting candidates for the treatment of AD. This review highlights the potential of specific compounds from natural products along with their synthetic derivatives to counteract AD in the CNS.

Potential for Treatment of Neurodegenerative Diseases with Natural Products or Synthetic Compounds that Stabilize Microtubules

2020 ◽  
Vol 26 (35) ◽  
pp. 4362-4372
Author(s):  
John H. Miller ◽  
Viswanath Das
Keyword(s):  
Natural Products ◽  
Neurodegenerative Diseases ◽  
In Vivo Models ◽  
Synthetic Compounds ◽  
Stabilizing Agents ◽  
Animal Models Of Disease ◽  
Model Studies ◽  
Models Of Disease

No effective therapeutics to treat neurodegenerative diseases exist, despite significant attempts to find drugs that can reduce or rescue the debilitating symptoms of tauopathies such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, or Pick’s disease. A number of in vitro and in vivo models exist for studying neurodegenerative diseases, including cell models employing induced-pluripotent stem cells, cerebral organoids, and animal models of disease. Recent research has focused on microtubulestabilizing agents, either natural products or synthetic compounds that can prevent the axonal destruction caused by tau protein pathologies. Although promising results have come from animal model studies using brainpenetrant natural product microtubule-stabilizing agents, such as paclitaxel analogs that can access the brain, epothilones B and D, and other synthetic compounds such as davunetide or the triazolopyrimidines, early clinical trials in humans have been disappointing. This review aims to summarize the research that has been carried out in this area and discuss the potential for the future development of an effective microtubule stabilizing drug to treat neurodegenerative disease.

Novel Phytochemical Constituents and their Potential to Manage Diabetes

2020 ◽  
Vol 26 ◽  
Author(s):  
Shaik Ibrahim Khalivulla ◽  
Arifullah Mohammed ◽  
Kokkanti Mallikarjuna
Keyword(s):  
Natural Products ◽  
Large Population ◽  
World Health ◽  
In Vivo Studies ◽  
Antidiabetic Activity ◽  
Therapeutic Drug ◽  
Pharmacological Activities ◽  
Chemical Structures

Background: Diabetes is a chronic disease affecting a large population worldwide and stands as one of the major global health challenges to be tackled. According to World Health Organization, about 400 million are having diabetes worldwide and it is the seventh leading cause of deaths in 2016. Plant based natural products had been in use from ancient time as ethnomedicine for the treatment of several diseases including diabetes. As a result of that, there are several reports on plant based natural products displaying antidiabetic activity. In the current review, such antidiabetic potential compounds reported from all plant sources along with their chemical structures are collected, presented and discussed. This kind of reports are essential to pool the available information to one source followed by statistical analysis and screening to check the efficacy of all known compounds in a comparative sense. This kind of analysis can give rise to few numbers of potential compounds from hundreds, whom can further be screened through in vitro and in vivo studies, and human trails leading to the drug development. Methods: Phytochemicals along with their potential antidiabetic property were classified according to their basic chemical skeleton. The chemical structures of all the compounds with antidiabetic activities were elucidated in the present review. In addition to this, the distribution and their other remarkable pharmacological activities of each species is also included. Results: The scrutiny of literature led to identification of 44 plants with antidiabetic compounds (70) and other pharmacological activities. For the sake of information, the distribution of each species in the world is given. Many plant derivatives may exert antidiabetic properties by improving or mimicking the insulin production or action. Different classes of compounds including sulfur compounds (1-4), alkaloids (5-11), phenolic compounds (12-17), tannins (18-23), phenylpropanoids (24-27), xanthanoids (28-31), amino acid (32), stilbenoid (33), benzofuran (34), coumarin (35), flavonoids (36-49) and terpenoids (50-70) were found to be active potential compounds for antidiabetic activity. Of the 70 listed compounds, majorly 17 compounds are from triterpenoids, 13 flavonoids and 7 are from alkaloids. Among all the 44 plant species, maximum number (7) of compounds are reported from Lagerstroemia speciosa followed by Momordica charantia (6) and S. oblonga with 5 compounds. Conclusion: This is the first paper to summarize the established chemical structures of phytochemicals that have been successfully screened for antidiabetic potential and their mechanisms of inhibition. The reported compounds could be considered as potential lead molecules for the treatment of type-2 diabetes. Further, molecular and clinical trials are required to select and establish the therapeutic drug candidates.

Modulation of Matrix Metalloproteinases by Plant-Derived Products

2020 ◽  
Vol 20 ◽  
Author(s):  
Nur Najmi Mohamad Anuar ◽  
Nurul Iman Natasya Zulkafali ◽  
Azizah Ugusman
Keyword(s):  
Clinical Trials ◽  
Natural Products ◽  
Matrix Metalloproteinases ◽  
Animal Studies ◽  
Current Knowledge ◽  
In Vitro Models ◽  
In Vivo Studies ◽  
Extracellular Matrix Components

: Matrix metalloproteinases (MMPs) are a group of zinc-dependent metallo-endopeptidase that are responsible towards the degradation, repair and remodelling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic and food industries. This review summarises the current knowledge on plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signalling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviours, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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