PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen ...levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell’s metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCFSkp2, which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.
•The prolyl-4-hydroxylase PHD1 is required for mitotic progression•PHD1 hydroxylates centrosomal protein Cep192 in vitro and in vivo•Hydroxylation of Cep192 at Pro1717 modulates Cep192 stability and function•Hydroxylation of Cep192 is required for E3 ligase SCFSkp2 binding to Cep192
Cellular oxygen is sensed by a class of dioxygenase enzymes including the prolyl hydroxylase PHD1. Moser et al. find that PHD1 acts directly on the centrosome protein Cep192 to induce its SCFSkp2-dependent ubiquitination and degradation. PHD1 thus links oxygen sensing to the cell-cycle control of centrosome duplication and ciliogenesis.
More than 70,000 different terpenoid structures are known so far; many of them offer highly interesting applications as pharmaceuticals, flavors and fragrances, or biofuels. Extraction of these ...compounds from their natural sources or chemical synthesis is—in many cases—technically challenging with low or moderate yields while wasting valuable resources. Microbial production of terpenoids offers a sustainable and environment-friendly alternative starting from simple carbon sources and, frequently, safeguards high product specificity. Here, we provide an overview on employing recombinant bacteria and yeasts for heterologous de novo production of terpenoids. Currently,
Escherichia coli
and
Saccharomyces cerevisiae
are the two best-established production hosts for terpenoids. An increasing number of studies have been successful in engineering alternative microorganisms for terpenoid biosynthesis, which we intend to highlight in this review. Moreover, we discuss the specific engineering challenges as well as recent advances for microbial production of different classes of terpenoids. Rationalizing the current stages of development for different terpenoid production hosts as well as future prospects shall provide a valuable decision basis for the selection and engineering of the cell factory(ies) for industrial production of terpenoid target molecules.
A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 ...(PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1's role and the mechanism regulating its function in the DCT.
We used
mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor-1 (I1), mediates cAMP's effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions.
Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation
a protein kinase A (PKA)-dependent pathway.
incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC
, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC.
Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation
a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.
In response to genotoxic insults, cells activate DNA damage response pathways that either stimulate transient cell cycle arrest and DNA repair or induce apoptosis. The Caenorhabditis elegans germ ...line is now well established as a model system to study these processes in a genetically tractable, multicellular organism. Upon treatment with genotoxic agents, premeiotic C. elegans germ cells transiently halt cell cycle progression, whereas meiotic prophase germ cells in the late-pachytene stage undergo apoptosis. Further, accumulation of unrepaired meiotic recombination intermediates can also lead to apoptosis of affected pachytene cells. DNA damage-induced cell death requires key components of the evolutionarily conserved apoptotic machinery. Moreover, both cell cycle arrest and pachytene apoptosis responses depend on conserved DNA damage checkpoint proteins. Genetics- and genomics-based approaches that have demonstrated roles for conserved checkpoint proteins have also begun to uncover novel components of these response pathways. In this chapter, we briefly review the C. elegans DNA damage response field, discuss in detail methods currently used to assay DNA damage responses in C. elegans, and describe the development of new experimental tools that will facilitate a more comprehensive understanding of the DNA damage response.
The triterpenoid (+)‐ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)‐ambrein, several fragrance ...molecules are formed, amongst them (−)‐ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole‐cell biocatalyst for the production of (+)‐ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N‐terminally truncated 3‐hydroxy‐3‐methylglutaryl‐CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl‐β‐curcumene cyclase (BmeTC‐D373C), which has been shown to be able to catalyse the conversion of squalene to 3‐deoxyachillol and then further to (+)‐ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole‐cell (+)‐ambrein production.
Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are ...structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.
CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, ...but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2-null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression ...through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.
SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ...ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways.
The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component ...of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L−1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein.
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•Squalene accumulation in P. pastoris for triterpenoid biosynthesis.•First whole-cell biosynthesis of (+)-ambrein in yeast.•Cell and enzyme engineering improved (+)-ambrein yield to> 100 mg L−1 culture.
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