14 research outputs found
Thiosuccinyl Peptides as Sirt5-Specific Inhibitors
Sirtuins, a class of enzymes known as nicotinamide adenine
dinucleotide-dependent
deacetylases, have been shown to regulate a variety of biological
processes, including aging, transcription, and metabolism. Sirtuins
are considered promising targets for treating several human diseases.
There are seven sirtuins in humans (Sirt1–7). Small molecules
that can target a particular human sirtuin are important for drug
development and fundamental studies of sirtuin biology. Here we demonstrate
that thiosuccinyl peptides are potent and selective Sirt5 inhibitors.
The design of these inhibitors is based on our recent discovery that
Sirt5 prefers to catalyze the hydrolysis of malonyl and succinyl groups,
rather than an acetyl group, from lysine residues. Furthermore, among
the seven human sirtuins, Sirt5 is the only one that has this unique
acyl group preference. This study demonstrates that the different
acyl group preferences of different sirtuins can be conveniently utilized
to develop small molecules that selectively target different sirtuins
Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors
Poly-ADP-ribosylation
is an important protein post-translational
modification with diverse biological consequences. After binding poly-ADP-ribose
on axis inhibition protein 1 (AXIN1) through its WWE domain, RING
finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its
proteasomal degradation and thus the oncogenic WNT signaling. Therefore,
inhibiting the RNF146 WWE domain is a potential antitumor strategy.
However, due to a lack of suitable screening methods, no inhibitors
for this domain have been reported. Here, we developed a fluorescence
polarization (FP)-based competition assay for the screening of RNF146
WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose
tracer compound, TAMRA-isoADPr. We report the design
and synthesis of this tracer compound and show that it is a high-affinity
tracer for the RNF146 WWE domain. This provides a convenient assay
and will facilitate the development of small-molecule inhibitors for
the RNF146 WWE domain
Iso-ADP-Ribose Fluorescence Polarization Probe for the Screening of RNF146 WWE Domain Inhibitors
Poly-ADP-ribosylation
is an important protein post-translational
modification with diverse biological consequences. After binding poly-ADP-ribose
on axis inhibition protein 1 (AXIN1) through its WWE domain, RING
finger protein 146 (RNF146) can ubiquitinate AXIN1 and promote its
proteasomal degradation and thus the oncogenic WNT signaling. Therefore,
inhibiting the RNF146 WWE domain is a potential antitumor strategy.
However, due to a lack of suitable screening methods, no inhibitors
for this domain have been reported. Here, we developed a fluorescence
polarization (FP)-based competition assay for the screening of RNF146
WWE inhibitors. This assay relies on a fluorescently tagged iso-ADP-ribose
tracer compound, TAMRA-isoADPr. We report the design
and synthesis of this tracer compound and show that it is a high-affinity
tracer for the RNF146 WWE domain. This provides a convenient assay
and will facilitate the development of small-molecule inhibitors for
the RNF146 WWE domain
Comparative Nucleotide-Dependent Interactome Analysis Reveals Shared and Differential Properties of KRas4a and KRas4b
The KRAS gene encodes
two isoforms, KRas4a and KRas4b. Differences
in the signaling functions of the two KRas proteins are poorly understood.
Here we report the comparative and nucleotide-dependent interactomes
of KRas4a and KRas4b. Many previously unknown interacting proteins
were identified, with some interacting with both isoforms while others
prefer only one. For example, v-ATPase a2 and eIF2Bδ interact
with only KRas4b. Consistent with the v-ATPase interaction, KRas4b
has a significant lysosomal localization. Comparing WT and constitutively
active G12D mutant KRas, we examined differences in the effector proteins
of the KRas4a and KRas4b. Interestingly, KRas4a binds RAF1 stronger
than KRas4b. Correspondingly, KRas4a can better promote ERK phosphorylation
and anchorage-independent growth than KRas4b. The interactome data
represent a useful resource to understand the differences between
KRas4a and KRas4b and to discover new function or regulation for them.
A similar proteomic approach would be useful for studying numerous
other small GTPases
Comparative Nucleotide-Dependent Interactome Analysis Reveals Shared and Differential Properties of KRas4a and KRas4b
The KRAS gene encodes
two isoforms, KRas4a and KRas4b. Differences
in the signaling functions of the two KRas proteins are poorly understood.
Here we report the comparative and nucleotide-dependent interactomes
of KRas4a and KRas4b. Many previously unknown interacting proteins
were identified, with some interacting with both isoforms while others
prefer only one. For example, v-ATPase a2 and eIF2Bδ interact
with only KRas4b. Consistent with the v-ATPase interaction, KRas4b
has a significant lysosomal localization. Comparing WT and constitutively
active G12D mutant KRas, we examined differences in the effector proteins
of the KRas4a and KRas4b. Interestingly, KRas4a binds RAF1 stronger
than KRas4b. Correspondingly, KRas4a can better promote ERK phosphorylation
and anchorage-independent growth than KRas4b. The interactome data
represent a useful resource to understand the differences between
KRas4a and KRas4b and to discover new function or regulation for them.
A similar proteomic approach would be useful for studying numerous
other small GTPases
Dph7 Catalyzes a Previously Unknown Demethylation Step in Diphthamide Biosynthesis
Present on archaeal
and eukaryotic translation elongation factor
2, diphthamide represents one of the most intriguing post-translational
modifications on proteins. The biosynthesis of diphthamide was proposed
to occur in three steps requiring seven proteins, Dph1–7, in
eukaryotes. The functional assignments of Dph1–5 in the first
and second step have been well established. Recent studies suggest
that Dph6 (yeast YLR143W or human ATPBD4) and Dph7 (yeast YBR246W
or human WDR85) are involved in the last amidation step, with Dph6
being the actual diphthamide synthetase catalyzing the ATP-dependent
amidation reaction. However, the exact molecular role of Dph7 is unclear.
Here we demonstrate that Dph7 is an enzyme catalyzing a previously
unknown step in the diphthamide biosynthesis pathway. This step is
between the Dph5- and Dph6-catalyzed reactions. We demonstrate that
the Dph5-catalyzed reaction generates methylated diphthine, a previously
overlooked intermediate, and Dph7 is a methylesterase that hydrolyzes
methylated diphthine to produce diphthine and allows the Dph6-catalyzed
amidation reaction to occur. Thus, our study characterizes the molecular
function of Dph7 for the first time and provides a revised diphthamide
biosynthesis pathway
SIRT2 Reverses 4‑Oxononanoyl Lysine Modification on Histones
Post-translational
modifications (PTMs) regulate numerous proteins
and are important for many biological processes. Lysine 4-oxononanoylation
(4-ONylation) is a newly discovered histone PTM that prevents nucleosome
assembly under oxidative stress. Whether there are cellular enzymes
that remove 4-ONyl from histones remains unknown, which hampers the
further investigation of the cellular function of this PTM. Here,
we report that mammalian SIRT2 can remove 4-ONyl from histones and
other proteins in live cells. A crystal structure of SIRT2 in complex
with a 4-ONyl peptide reveals a lone pair−π interaction
between Phe119 and the ketone oxygen of the 4-ONyl group. This is
the first time that a mechanism to reverse 4-ONyl lysine modification
is reported and will help to understand the role of SIRT2 in oxidative
stress responses and the function of 4-ONylation
YBR246W Is Required for the Third Step of Diphthamide Biosynthesis
Diphthamide, the target of diphtheria toxin, is a post-translationally
modified histidine residue that is found in archaeal and eukaryotic
translation elongation factor 2. The biosynthesis and function of
this modification has attracted the interest of many biochemists for
decades. The biosynthesis has been known to proceed in three steps.
Proteins required for the first and second steps have been identified,
but the proteinÂ(s) required for the last step have remained elusive.
Here we demonstrate that the YBR246W gene in yeast is required for
the last step of diphthamide biosynthesis, as the deletion of YBR246W
leads to the accumulation of diphthine, which is the enzymatic product
of the second step of the biosynthesis. This discovery will provide
important information leading to the complete elucidation of the full
biosynthesis pathway of diphthamide
<i>Plasmodium falciparum</i> Sir2A Preferentially Hydrolyzes Medium and Long Chain Fatty Acyl Lysine
<i>Plasmodium falciparum</i> Sir2A (PfSir2A),
a member
of the sirtuin family of nicotinamide adenine dinucleotide-dependent
deacetylases, has been shown to regulate the expression of surface
antigens to evade the detection by host immune surveillance. It is
thought that PfSir2A achieves this by deacetylating histones. However,
the deacetylase activity of PfSir2A is weak. Here we present enzymology
and structural evidence supporting that PfSir2A catalyzes the hydrolysis
of medium and long chain fatty acyl groups from lysine residues more
efficiently. Furthermore, <i>P. falciparum</i> proteins
are found to contain such fatty acyl lysine modifications that can
be removed by purified PfSir2A <i>in vitro</i>. Together,
the data suggest that the physiological function of PfSir2A in antigen
variation may be achieved by removing medium and long chain fatty
acyl groups from protein lysine residues. The robust activity of PfSir2A
would also facilitate the development of PfSir2A inhibitors, which
may have therapeutic value in malaria treatment
HDAC8 Catalyzes the Hydrolysis of Long Chain Fatty Acyl Lysine
The histone deacetylase (HDAC) family
regulates many biological
pathways through the deacetylation of lysine residues on histone and
nonhistone proteins. Mammals have 18 HDACs that are classified into
four classes. Class I, II, and IV are zinc-dependent, while class
III is nicotinamide adenine dinucleotide (NAD<sup>+</sup>)-dependent
lysine deacetylase or sirtuins. HDAC8, a class I HDAC family member,
has been shown to have low deacetylation activity compared to other
HDACs <i>in vitro.</i> Recent studies showed that several
sirtuins, with low deacetylase activities, can actually hydrolyze
other acyl lysine modifications more efficiently. Inspired by this,
we tested the activity of HDAC8 using a variety of different acyl
lysine peptides. Screening a panel of peptides with different acyl
lysine modifications, we found that HDAC8 can catalyze the removal
of acyl groups with 2–16 carbons from lysine 9 of the histone
H3 peptide (H3K9). Interestingly, the catalytic efficiencies (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) of HDAC8
on octanoyl, dodecanoyl, and myristoyl lysine are several-fold better
than that on acetyl lysine. The increased catalytic efficiencies of
HDAC8 on larger fatty acyl groups are due to the much lower <i>K</i><sub>m</sub> values. T-cell leukemia Jurkat cells treated
with a HDAC8 specific inhibitor, PCI-34051, exhibited an increase
in global fatty acylation compared to control treatment. Thus, the
de-fatty-acylation activity of HDAC8 is likely physiologically relevant.
This is the first report of a zinc-dependent HDAC with de-fatty-acylation
activity, and identification of HDAC8 de-fatty-acylation targets will
help to further understand the function of HDAC8 and protein lysine
fatty acylation