65 research outputs found
Identification of a Novel Class of Farnesylation Targets by Structure-Based Modeling of Binding Specificity
Farnesylation is an important post-translational modification catalyzed by farnesyltransferase (FTase). Until recently it was believed that a C-terminal CaaX motif is required for farnesylation, but recent experiments have revealed larger substrate diversity. In this study, we propose a general structural modeling scheme to account for peptide binding specificity and recapitulate the experimentally derived selectivity profile of FTase in vitro. In addition to highly accurate recovery of known FTase targets, we also identify a range of novel potential targets in the human genome, including a new substrate class with an acidic C-terminal residue (CxxD/E). In vitro experiments verified farnesylation of 26/29 tested peptides, including both novel human targets, as well as peptides predicted to tightly bind FTase. This study extends the putative range of biological farnesylation substrates. Moreover, it suggests that the ability of a peptide to bind FTase is a main determinant for the farnesylation reaction. Finally, simple adaptation of our approach can contribute to more accurate and complete elucidation of peptide-mediated interactions and modifications in the cell
Maternal smoking during pregnancy and birth defects in children: a systematic review with meta-analysis
Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates
Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases
Site-specific
protein labeling is an important technique in protein
chemistry and is used for diverse applications ranging from creating
protein conjugates to protein immobilization. Enzymatic reactions,
including protein prenylation, have been widely exploited as methods
to accomplish site-specific labeling. Enzymatic prenylation is catalyzed
by prenyltransferases, including protein farnesyltransferase (PFTase)
and geranylgeranyltransferase type I (GGTase-I), both of which recognize
C-terminal CaaX motifs with different specificities and transfer prenyl
groups from isoprenoid diphosphates to their respective target proteins.
A number of isoprenoid analogues containing bioorthogonal functional
groups have been used to label proteins of interest via PFTase-catalyzed
reaction. In this study, we sought to expand the scope of prenyltransferase-mediated
protein labeling by exploring the utility of rat GGTase-I (rGGTase-I).
First, the isoprenoid specificity of rGGTase-I was evaluated by screening
eight different analogues and it was found that those with bulky moieties
and longer backbone length were recognized by rGGTase-I more efficiently.
Taking advantage of the different substrate specificities of rat PFTase
(rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling
method to selectively label two different proteins by using isoprenoid
analogue and CaaX substrate pairs that were specific to only one of
the prenyltransferases. Using two model proteins, green fluorescent
protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent
protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated
that when incubated together with both prenyltransferases and the
selected isoprenoid analogues, GFP-CVLL was specifically modified
with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was
selectively labeled with an alkyne-containing analogue by rPFTase.
By switching the ketone-containing analogue to an azide-containing
analogue, it was possible to create protein tail-to-tail dimers in
a one-pot procedure through the copperÂ(I)-catalyzed alkyne–azide
cycloaddition (CuAAC) reaction. Overall, with the flexibility of using
different isoprenoid analogues, this system greatly extends the utility
of protein labeling using prenyltransferases
Targeted Reengineering of Protein Geranylgeranyltransferase Type I Selectivity Functionally Implicates Active-Site Residues in Protein-Substrate Recognition
Adverse drug events in German hospital routine data: A validation of International Classification of Diseases, 10th revision (ICD-10) diagnostic codes
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