6 research outputs found
Precursor-Directed Mutational Biosynthesis Facilitates the Functional Assignment of Two Cytochromes P450 in Thiostrepton Biosynthesis
Side-ring-modified thiostrepton (TSR)
derivatives that vary in their quinaldic acid (QA) substitution possess
more potent biological activities and better pharmaceutical properties
than the parent compound. In this work, we sought to introduce fluorine
onto C-7′ or C-8′ of the TSR QA moiety via precursor-directed
mutational biosynthesis to obtain new TSR variants. Unexpectedly,
instead of the target product, the exogenous chemical feeding of 7-F-QA
into the Δ<i>tsrT</i> mutant strain resulted in a
unique TSR analog with an incomplete side-ring structure and an unoxidized
QA moiety (<b>1</b>). Accordingly, two cytochrome P450 genes, <i>tsrP</i> and <i>tsrR</i>, were in-frame deleted to
elucidate the candidate responsible for the monooxidation of the QA
moiety in TSR. The unfluorinated analog of compound <b>1</b> that was thus isolated from Δ<i>tsrP</i> (<b>2</b>) and the abolishment of TSR production in Δ<i>tsrR</i> revealed not only the biosynthetic logic of the TSR
side-ring but also the essential checkpoint in TSR maturation before
macro-ring closure
Thiolation Protein-Based Transfer of Indolyl to a Ribosomally Synthesized Polythiazolyl Peptide Intermediate during the Biosynthesis of the Side-Ring System of Nosiheptide
Nosiheptide,
a potent bicyclic member of the family of thioÂpeptide
antibiotics, possesses a distinctive l-Trp-derived indolyl
moiety. The way in which this moiety is incorporated into a ribosomally
synthesized and post-translationally modified thioÂpeptide remains
poorly understood. Here, we report that NosK, an α/β-hydrolase
fold protein, mediates the transfer of indolyl from NosJ, a discrete
thiolation protein, to a linear pentathiazolyl peptide intermediate
rather than its genetically encoded untreated precursor. This intermediate
results from enzymatic processing of the peptide precursor, in which
five of the six l-Cys residues are transformed into thiazoles
but Cys4 selectively remains unmodified for indolyl substitution via
a thioester exchange. Determining the timing of indolyl incorporation,
which expands the chemical space of a thioÂpeptide framework,
facilitates mechanistic access to the unusual logic of post-translational
modifications in the biosynthesis of nosiÂheptide-type thioÂpeptide
members that share a similar compact side-ring system
Specific and Efficient N‑Propionylation of Histones with Propionic Acid <i>N</i>‑Hydroxysuccinimide Ester for Histone Marks Characterization by LC-MS
Histones participate in epigenetic regulation via a variety
of
dynamic posttranslational modifications (PTMs) on them. Mass spectrometry
(MS) has become a powerful tool to investigate histone PTMs. With
the bottom-up mass spectrometry approach, chemical derivatization
of histones with propionic anhydride or deuterated acetic anhydride
followed by trypsin digestion was widely used to block the hydrophilic
lysine residues and generate compatible peptides for LC-MS analysis.
However, certain severe side reactions (such as acylation on tyrosine
or serine) caused by acid anhydrides will lead to a number of analytical
issues such as reducing results accuracy and impairing the reproducibility
and sensitivity of MS analysis. As an alternative approach, we report
a novel derivatization method that utilizes <i>N</i>-hydroxysuccinimide
ester to specifically and efficiently derivatize both free and monomethylated
amine groups in histones. A competitive inhibiting strategy was implemented
in our method to effectively prevent the side reactions. We demonstrated
that our method can achieve excellent specificity and efficiency for
histones derivatization in a reproducible manner. Using this derivatization
method, we succeeded to quantitatively profile the histone PTMs in
KMS11 cell line with selective knock out of translocated NSD2 allele
(TKO) and the original parental KMS11 cell lines (PAR) (NSD2, a histone
methyltransferase that catalyzes the histone H3 K36 methylation),
which revealed a significant crosstalk between H3 protein K27 methylation
and adjacent K36 methylation
Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS
Deciphering
the combinatorial histone codes has been a long-standing
interest in the epigenetics field, which requires the reliable and
robust characterization of the post-translational modifications (PTMs)
coexisting on histones. To this end, weak cation exchange hydrophilic
interaction liquid chromatography is commonly used in middle-down
liquid chromatography–mass spectrometry approaches for online
separation of variously modified histone peptides. Here we provide
a novel strategy that combines the selective histone peptide derivatization
using <i>N</i>-hydroxysuccinimide propionate ester with
reversed-phase liquid chromatography (RPLC) for the robust, sensitive,
and reliable characterization of combinatorial histone PTMs. Derivatization
amplifies the subtle physical differences between similarly modified
histone peptides, thereby allowing baseline separation of these peptides
by standard RPLC. Also, the sensitivity of MS is enhanced greatly
by derivatization due to the increased peptide hydrophobicity and
concentrated charge-state envelope during electrospray ionization.
Furthermore, we systematically optimized the dual electron transfer
and higher energy collision dissociation and achieved near-complete
peptide sequence coverage in MS/MS spectra, allowing highly precise
and reliable PTM identification. Using this method, we identified
311 and 293 combinations of histone H3 PTMs from the lymphoma cells
Karpas-422 with/without drug treatment, confirming the advantages
of our method in serving as a platform for profiling combinatorial
histone PTMs
Quantitative Profiling of Combinational K27/K36 Modifications on Histone H3 Variants in Mouse Organs
The
coexisting post-translational modifications (PTMs) on histone
H3 N-terminal tails were known to crosstalk between each other, indicating
their interdependency in the epigenetic regulation pathways. H3K36
methylation, an important activating mark, was recently reported to
antagonize with PRC2-mediated H3K27 methylation with possible crosstalk
mechanism during transcription regulation process. On the basis of our previous studies, we further integrated
RP/HILIC liquid chromatography with MRM mass spectrometry to quantify
histone PTMs from various mouse organs, especially the combinatorial
K27/K36 marks for all three major histone H3 variants. Despite their
subtle difference in physicochemical properties, we successfully obtained
decent separation and high detection sensitivity for both histone
H3.3 specific peptides and histone H3.1/3.2 specific peptides. In
addition, the overall abundance of H3.3 can be quantified simultaneously.
We applied this method to investigate the pattern of the combinatorial
K27/K36 marks for all three major histone H3 variants across five
mouse organs. Intriguing distribution differences were observed not
only between different H3 variants but also between different organs.
Our data shed the new insights into histone codes functions in epigenetic
regulation during cell differentiation and developmental process
Absolute Quantification of Histone PTM Marks by MRM-Based LC-MS/MS
The N-terminal tails of core histones
harbor the sites of numerous
post-translational modifications (PTMs) with important roles in the
regulation of chromatin structure and function. Profiling histone
PTM marks provides data that help understand the epigenetics events
in cells and their connections with cancer and other diseases. Our
previous study demonstrated that specific derivatization of histone
peptides by NHS propionate significantly improved their chromatographic
performance on reversed phase columns for LC/MS analysis. As a step
forward, we recently developed a multiple reaction monitoring (MRM)
based LC-MS/MS method to analyze 42 targeted histone peptides. By
using stable isotopic labeled peptides as internal standards that
are spiked into the reconstituted solutions, this method allows to
measure absolute concentration of the tryptic peptides of H3 histone
proteins extracted from cancer cell lines. The method was thoroughly
validated for the accuracy and reproducibility through analyzing recombinant
histone proteins and cellular samples. The linear dynamic range of
the MRM assays was achieved in 3 orders of magnitude from 1 nM to
1 ÎĽM for all targeted peptides. Excellent intrabatch and interbatch
reproducibility (<15% CV) was obtained. This method has been used
to study translocated NSD2 (a histone lysine methyltransferase that
catalyzes the histone lysine 36 methylation) function with its overexpression
in KMS11 multiple myeloma cells. From the results we have successfully
quantitated both individual and combinatorial histone marks in parental
and NSD2 selective knockout KMS11 cells