4 research outputs found
Synthesis of Dimeric ADP-Ribose and Its Structure with Human Poly(ADP-ribose) Glycohydrolase
Poly(ADP-ribosyl)ation
is a common post-translational modification
that mediates a wide variety of cellular processes including DNA damage
repair, chromatin regulation, transcription, and apoptosis. The difficulty
associated with accessing poly(ADP-ribose) (PAR) in a homogeneous
form has been an impediment to understanding the interactions of PAR
with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins.
Here we describe the chemical synthesis of the ADP-ribose dimer, and
we use this compound to obtain the first human PARG substrate-enzyme
cocrystal structure. Chemical synthesis of PAR is an attractive alternative
to traditional enzymatic synthesis and fractionation, allowing access
to products such as dimeric ADP-ribose, which has been detected but
never isolated from natural sources. Additionally, we describe the
synthesis of an alkynylated dimer and demonstrate that this compound
can be used to synthesize PAR probes including biotin and fluorophore-labeled
compounds. The fluorescently labeled ADP-ribose dimer was then utilized
in a general fluorescence polarization-based PAR–protein binding
assay. Finally, we use intermediates of our synthesis to access various
PAR fragments, and evaluation of these compounds as substrates for
PARG reveals the minimal features for substrate recognition and enzymatic
cleavage. Homogeneous PAR oligomers and unnatural variants produced
from chemical synthesis will allow for further detailed structural
and biochemical studies on the interaction of PAR with its many protein
binding partners
Synthetic α- and β‑Ser-ADP-ribosylated Peptides Reveal α‑Ser-ADPr as the Native Epimer
A solid-phase methodology to synthesize
oligopeptides, specifically
incorporating serine residues linked to ADP-ribose (ADPr), is presented.
Through the synthesis of both α- and β-anomers of the
phosphoribosylated Fmoc-Ser building block and their usage in our
modified solid-phase peptide synthesis protocol, both α- and
β-ADPr peptides from a naturally Ser-ADPr containing H2B sequence
were obtained. With these, and by digestion studies using the human
glycohydrolase, ARH3 (hARH3), compelling evidence is obtained that
the α-Ser-ADPr linkage comprises the naturally occurring configuration
Four of a Kind: A Complete Collection of ADP-Ribosylated Histidine Isosteres Using Cu(I)- and Ru(II)-Catalyzed Click Chemistry
Adenosine diphosphate
ribosylation (ADP-ribosylation) is a crucial
post-translational modification involved in important regulatory mechanisms
of numerous cellular pathways including histone maintenance and DNA
damage repair. To study this modification, well-defined ADP-ribosylated
peptides, proteins, and close analogues thereof have been invaluable
tools. Recently, proteomics studies have revealed histidine residues
to be ADP-ribosylated. We describe here the synthesis of a complete
set of triazole-isosteres of ADP-ribosylated histidine to serve as
probes for ADP-ribosylating biomachinery. By exploiting Cu(I)- and
Ru(II)-catalyzed click chemistry between a propargylglycine building
block and an α- or β-configured azidoribose, we have successfully
assembled the α- and β-configured 1,4- and 1,5-triazoles,
mimicking N(τ)- and N(π)-ADP-ribosylated histidine, respectively.
The ribosylated building blocks could be incorporated into a peptide
sequence using standard solid-phase peptide synthesis and transformed
on resin into the ADP-ribosylated fragments to provide a total of
four ADP-ribosyl triazole conjugates, which were evaluated for their
chemical and enzymatic stability. The 1,5-triazole analogues mimicking
the N(π)-substituted histidines proved susceptible to base-induced
epimerization and the ADP-ribosyl α-1,5-triazole linkage could
be cleaved by the (ADP-ribosyl)hydrolase ARH3
Discovery of a Selective Allosteric Inhibitor Targeting Macrodomain 2 of Polyadenosine-Diphosphate-Ribose Polymerase 14
Macrodomains
are conserved protein interaction modules that can
be found in all domains of life including in certain viruses. Macrodomains
mediate recognition of sequence motifs harboring adenosine diphosphate
ribose (ADPR) modifications, thereby regulating a variety of cellular
processes. Due to their role in cancer or viral pathogenesis, macrodomains
have emerged as potential therapeutic targets, but the unavailability
of small molecule inhibitors has hampered target validation studies
so far. Here, we describe an efficient screening strategy for identification
of small molecule inhibitors that displace ADPR from macrodomains.
We report the discovery and characterization of a macrodomain inhibitor,
GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low
micromolar affinity. Co-crystallization of a GeA-69 analogue with
PARP14 MD2 revealed an allosteric binding mechanism explaining its
selectivity over other human macrodomains. We show that GeA-69 engages
PARP14 MD2 in intact cells and prevents its localization to sites
of DNA damage