Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

Twining, Sally S.; Goryshin, Igor Y.; Bhasin, Archna; Reznikoff, William S.

doi:10.1074/jbc.m010748200

Cited by 17 publications

(17 citation statements)

References 26 publications

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“…A six-histidine tag with in-frame stop codon was introduced upstream of the glycosylphosphatidylinositol anchor site at amino acid 338 and transformed into yeast strain BJ3505. Protein expression and purification were then performed as described previously (13).…”

Section: S4mentioning

confidence: 99%

β2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes

Scott

Billiar

2008

Journal of Biological Chemistry

113

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The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the ␤2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.The liver is the first major organ downstream of the gut and is responsible for the clearance and initial recognition of the majority of LPS 2 (1). Studies using radiolabeled LPS characterized the uptake and processing of LPS within the liver more than 20 years ago (2, 3). LPS is taken up by both parenchymal cells (hepatocytes) and nonparenchymal cells (Kupffer cells, hepatic stellate cells, etc.) in the liver, and it appears in the bile within 15 min of intravenous injection (4). Controversy remains regarding the relative roles of Kupffer cells and hepatocytes in the uptake and clearance of LPS. Kupffer cells rapidly produce a strong cytokine response to LPS (5) and can deacylate LPS using a lipase (6). Another study suggested that some types of LPS are preferentially taken up by Kupffer cells and deacylated, which then allows for more rapid internalization within hepatocytes (7). However other studies using gadolinium chloride to deplete Kupffer cells have determined that hepatocytes also play a major role in LPS clearance (4). Regardless, it is clear that hepatocytes, which form the largest mass of cells in the liver and are constantly exposed to endotoxin from the gut, play an important role in LPS uptake, and innate immune responses to LPS are dependent on this process.The mechanisms involved in the internalization of LPS remain poorly understood, however. Our laboratory has previously shown that hepatocytes express the cell surface components of the LPS receptor/signaling complex (CD14/TLR4/ MD2) (8 -10) and that signaling activated by LPS through this receptor complex on hepatocytes leads to the activation of MAPK signaling proteins, translocation of NFB to the nucleus, and also production and release of acute phase proteins such as soluble CD14 and LPS-binding protein (8, 9). However, the functional role of the LPS recognition complex on hepatocytes remains uncertain. Here we hypothesize that this complex plays a role in the binding and initiation of uptake of LPS into cells.In this study we have clarified the mechanism of initial uptake of LPS into primary isolated hepatocytes. As expected, we found that CD14, MD2, and TLR4 were requ...

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Section: S4mentioning

confidence: 99%

β2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes

Scott

Billiar

2008

Journal of Biological Chemistry

113

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“…The in vitro cleavage of Mos1 adjacent to Arg119, Arg167 and Arg183 is consistent with the action of a protease and may re¯ect a role for protein degradation in vivo. For Tn5 it has been suggested that disengagement of the protein from DNA after transposition may be facilitated in vivo by proteolytic cleavage at Lys40 (Twining et al, 2001). …”

Section: Biochemical Analysis Of the Crystalsmentioning

confidence: 99%

Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

Richardson

Zhang

Marcos

et al. 2004

Acta Crystallogr D Biol Cryst

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A soluble single-point mutant of full-length Mos1 mariner transposase (MW = 40.7 kDa) has been overexpressed in Escherichia coli, puri®ed to 95% homogeneity and crystallized. This provides the ®rst example of the crystallization of a eukaryotic transposase. The native crystals diffract to 2.5 A Ê resolution and show tetragonal symmetry, with unit-cell parameters a = b = 44.5, c = 205.6 A Ê . Multiplewavelength anomalous data from a selenomethionyl form of the protein and data from a heavy-atom derivative have been collected.

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“…2 This combination of Tnp EK/LP and ME-flanked DNAs has resulted in the development of an efficient series of gel shift assays (4) and catalytic assays (2,23) used to study the transposition process. Tn5 in vitro assays have also been used to study C-terminally truncated forms of Tnp EK/LP and fulllength Tnp EK/LP with point mutations added to either the N or C terminus (26,27).…”

Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn 5 Transposase Active Site Mutants

Naumann

Reznikoff

2002

Journal of Biological Chemistry

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Tn5 transposase (Tnp) is a 53.3-kDa protein that is encoded by and facilitates movement of transposon Tn5. Tnp monomers contain a single active site that is responsible for catalyzing a series of four DNA breaking/ joining reactions at one transposon end. Based on primary sequence homology and protein structural information, we designed and constructed a series of plasmids that encode for Tnps containing active site mutations. Following Tnp expression and purification, the active site mutants were tested for their ability to form protein-DNA complexes and perform each of the four catalytic steps in the transposition pathway in vitro. The results demonstrate that Asp-97, Asp-188, and Glu-326, visible in the active site of Tn5 crystal structures, are absolutely required for all catalytic steps. Mutations within a series of amino acid residues that are conserved in the IS4 family of transposases and retroviral integrases also impair Tnp catalytic activity. Mutations at either Tyr-319 or Arg-322 reduce both hairpin resolution and strand transfer activity within protein-DNA complexes. Mutations at Lys-333 reduce the ability of Tnps to form protein-DNA complexes, whereas mutations at the less strongly conserved Lys-330 have less of an effect on both synaptic complex formation and catalytic activity.

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Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

Cited by 17 publications

References 26 publications

β2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes

β2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes

Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

Tn 5 Transposase Active Site Mutants

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