scholarly journals In vitro characterization of the colibactin-activating peptidase ClbP enables development of a fluorogenic activity probe

2019 ◽  
Author(s):  
Matthew R. Volpe ◽  
Matthew R. Wilson ◽  
Carolyn A. Brotherton ◽  
Ethan S. Winter ◽  
Sheila E. Johnson ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inner Membrane ◽  
Substrate Preferences ◽  
In Vitro Characterization ◽  
Catalytically Active ◽  
Membrane Bound

ABSTRACTThe gut bacterial genotoxin colibactin has been linked to the development of colorectal cancer. In the final stages of colibactin’s biosynthesis, an inactive precursor (pre-colibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

Biochemical Characterization of Chloromethane Emission from the Wood-Rotting Fungus Phellinus pomaceus

1998 ◽  
Vol 64 (8) ◽  
pp. 2831-2835 ◽  
Author(s):  
Deepti Saxena ◽  
Saleh Aouad ◽  
Jihad Attieh ◽  
Hargurdeep S. Saini
Keyword(s):  
Atmospheric Chemistry ◽  
Biochemical Characterization ◽  
Active Form ◽  
Methyl Chloride ◽  
Bound State ◽  
Ph Optimum ◽  
Methyl Donors ◽  
Membrane Bound

ABSTRACT Many wood-rotting fungi, including Phellinus pomaceus, produce chloromethane (CH3Cl). P. pomaceus can be cultured in undisturbed glucose mycological peptone liquid medium to produce high amounts of CH3Cl. The biosynthesis of CH3Cl is catalyzed by a methyl chloride transferase (MCT), which appears to be membrane bound. The enzyme is labile upon removal from its natural location and upon storage at low temperature in its bound state. Various detergents failed to solubilize the enzyme in active form, and hence it was characterized by using a membrane fraction. The enzyme had a sharp pH optimum between 7 and 7.2. Its apparent Km for Cl− (ca. 300 mM) was much higher than that for I− (250 μM) or Br− (11 mM). A comparison of theseKm values to the relative in vivo methylation rates for different halides suggests that the realKm for Cl− may be much lower, but the calculated value is high because the CH3Cl produced is used immediately in a coupled reaction. Among various methyl donors tested, S-adenosyl-l-methionine (SAM) was the only one that supported significant methylation by MCT. The reaction was inhibited by S-adenosyl-l-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings advance our comprehension of a poorly understood metabolic sector at the origin of biogenic emissions of halomethanes, which play an important role in atmospheric chemistry.

scholarly journals The morphological and biochemical characterization of a line of rat promegakaryoblasts

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 110-121
Author(s):  
R Weinstein ◽  
MB Stemerman ◽  
DE MacIntyre ◽  
HN Steinberg ◽  
T Maciag
Keyword(s):  
Biochemical Characterization ◽  
Stage Iv ◽  
In Vitro Study ◽  
Stage Iii ◽  
Immunofluorescent Staining ◽  
Morphological Evidence ◽  
Specific Proteins ◽  
Membrane Bound

Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

scholarly journals The morphological and biochemical characterization of a line of rat promegakaryoblasts

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 110-121 ◽  
Author(s):  
R Weinstein ◽  
MB Stemerman ◽  
DE MacIntyre ◽  
HN Steinberg ◽  
T Maciag
Keyword(s):  
Biochemical Characterization ◽  
Stage Iv ◽  
In Vitro Study ◽  
Stage Iii ◽  
Immunofluorescent Staining ◽  
Morphological Evidence ◽  
Specific Proteins ◽  
Membrane Bound

Abstract Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

scholarly journals Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mikołaj Piotr Zaborowski ◽  
Pike See Cheah ◽  
Xuan Zhang ◽  
Isabella Bushko ◽  
Kyungheon Lee ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Extracellular Vesicles ◽  
Active Form ◽  
Ectodomain Shedding ◽  
Protein Cleavage ◽  
Gaussia Luciferase ◽  
Membrane Bound

AbstractExtracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

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