5 research outputs found
Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses
Posttranslational
modifications (PTMs) are the lingua franca of
cellular communication. Most PTMs are enzyme-orchestrated. However,
the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed
electrophile-signaling pathways. Despite the latest impetus toward
harnessing kinetically and functionally privileged cysteines for electrophilic
drug design, identifying these sensors remains challenging. Herein,
we designed “G-REX”a technique that allows controlled
release of reactive electrophiles in vivo. Mitigating toxicity/off-target
effects associated with uncontrolled bolus exposure, G-REX tagged <i>first-responding</i> innate cysteines that bind electrophiles
under true <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> conditions. G-REX identified two allosteric ubiquitin-conjugating
proteinsUbe2V1/Ube2V2sharing a novel privileged-sensor-cysteine.
This non-enzyme-catalyzed-PTM triggered responses <i>specific
to each</i> protein. Thus, G-REX is an unbiased method to identify
novel functional cysteines. Contrasting conventional active-site/off-active-site
cysteine-modifications that regulate <i>target</i> activity,
modification of Ube2V2 allosterically hyperactivated its enzymatically
active binding-partner Ube2N, promoting K63-linked client ubiquitination
and stimulating H2AX-dependent DNA damage response. This work establishes
Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard
genome integrity
Temporally Controlled Targeting of 4‑Hydroxynonenal to Specific Proteins in Living Cells
In-depth chemical understanding of
complex biological processes
hinges upon the ability to systematically perturb individual systems.
However, current approaches to study impacts of biologically relevant
reactive small molecules involve bathing of the entire cell or isolated
organelle with excess amounts, leading to off-target effects. The
resultant lack of biochemical specificity has plagued our understanding
of how biological electrophiles mediate signal transduction or regulate
responses that confer defense mechanisms to cellular electrophilic
stress. Here we introduce a target-specific electrophile delivery
platform that will ultimately pave the way to interrogate effects
of reactive electrophiles on specific target proteins in cells. The
new methodology is demonstrated by photoinducible targeted delivery
of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent
conjugation of the HNE-precursor to HaloTag fused to the target proteins
enables directed HNE delivery upon photoactivation. The strategy provides
proof of concept of selective delivery of reactive electrophiles to
individual electrophile-responsive proteins in mammalian cells. It
opens a new avenue enabling more precise determination of the pathophysiological
consequences of HNE-induced chemical modifications on specific target
proteins in cells
Cardiovascular Small Heat Shock Protein HSPB7 Is a Kinetically Privileged Reactive Electrophilic Species (RES) Sensor
Small
heat shock
protein (sHSP)-B7 (HSPB7) is a muscle-specific
member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is
enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically
privileged sensor that is able to react rapidly with native reactive
electrophilic species (RES), when only substoichiometric amounts of
RES are available in proximity to Hspb7 expressed in living cells.
Among the two Hspb7-cysteines, this RES sensing is fulfilled by a
single cysteine (C117). Purification and characterizations <i>in vitro</i> reveal that the rate for RES adduction is among
the most efficient reported for protein-cysteines with native carbonyl-based
RES. Covalent-ligand binding is accompanied by structural changes
(increase in β-sheet-content), based on circular dichroism analysis.
Among the two cysteines, only C117 is conserved across vertebrates;
we show that the human ortholog is also capable of RES sensing in
cells. Furthermore, a cancer-relevant missense mutation reduces this
RES-sensing property. This evolutionarily conserved cysteine-biosensor
may play a redox-regulatory role in cardioprotection
Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition
The enzyme ribonucleotide reductase
(RNR) is a major target of
anticancer drugs. Until recently, suicide inactivation in which synthetic
substrate analogs (nucleoside diphosphates) irreversibly inactivate
the RNR-α<sub>2</sub>β<sub>2</sub> heterodimeric complex
was the only clinically proven inhibition pathway. For instance, this
mechanism is deployed by the multifactorial anticancer agent gemcitabine
diphosphate. Recently reversible targeting of RNR-α-alone coupled
with ligand-induced RNR-α-persistent hexamerization has emerged
to be of clinical significance. To date, clofarabine nucleotides are
the only known example of this mechanism. Herein, chemoenzymatic syntheses
of the active forms of two other drugs, phosphorylated cladribine
(ClA) and fludarabine (FlU), allow us to establish that reversible
inhibition is common to numerous drugs in clinical use. Enzyme inhibition
and fluorescence anisotropy assays show that the di- and triphosphates
of the two nucleosides function as reversible (i.e., nonmechanism-based)
inhibitors of RNR and interact with the catalytic (C site) and the
allosteric activity (A site) sites of RNR-α, respectively. Gel
filtration, protease digestion, and FRET assays demonstrate that inhibition
is coupled with formation of conformationally diverse hexamers. Studies
in 293T cells capable of selectively inducing either wild-type or
oligomerization-defective mutant RNR-α overexpression delineate
the central role of RNR-α oligomerization in drug activity,
and highlight a potential resistance mechanism to these drugs. These
data set the stage for new interventions targeting RNR oligomeric
regulation
Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole-Cell-Stimulated Antioxidant Response
Lipid-derived
electrophiles (LDEs) that can directly modify proteins
have emerged as important small-molecule cues in cellular decision-making.
However, because these diffusible LDEs can modify many targets [e.g.,
>700 cysteines are modified by the well-known LDE 4-hydroxynonenal
(HNE)], establishing the functional consequences of LDE modification
on individual targets remains devilishly difficult. Whether LDE modifications
on a single protein are biologically sufficient to activate discrete
redox signaling response downstream also remains untested. Herein,
using T-REX (targetable reactive electrophiles and oxidants), an approach
aimed at selectively flipping a single redox switch in cells at a
precise time, we show that a modest level (∼34%) of HNEylation
on a single target is sufficient to elicit the pharmaceutically important
antioxidant response element (ARE) activation, and the resultant strength
of ARE induction recapitulates that observed from whole-cell electrophilic
perturbation. These data provide the first evidence that single-target
LDE modifications are important individual events in mammalian physiology