top of page
Publications: CV

Reprogramming a Deubiquitinase into a Transamidase

Lin Hui Chang and Eric R. Strieter (2018) ACS Chem. Biol. 13, 2808-2818, DOI: 10.1021/acschembio.8b00759.

Using protein engineering strategies we transform a DUB into a transamidase capable of modifying itself with defined Ub chains. The resulting conjugates can be used to investigate how DUBs and Ub-binding domains respond to a chain when it is anchored to a substrate protein.

DUB-to-Ligase ACS ChemBio TOC-01.jpg

Anal. Biochem. 2018

We developed a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method is based on a fluorescence polarization competition (FPcomp) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains.


Anal. Biochem. 2018


Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains

J. Proteome Res. 2017

By combining a Lys11 linkage-specific antibody with high-resolution middle-down mass spectrometry (an approach termed UbiChEM-MS) we identify endogenous Lys11/Lys48 branched ubiquitin chains in cells.


Determining Atomistic SAXS Models of Tri-Ubiquitin Chains from Bayesian Analysis of Accelerated Molecular Dynamics Simulations

J. Chem. Theory. Comput. 2017

Here, we demonstrate that accelerated MD simulations can be used to produce higher quality models in shorter time scales than standard simulations, and we present an iterative Bayesian Monte Carlo method that is able to identify multistate ensembles without overfitting.


Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo

Anal. Chem. 2017

Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods.


Selenocysteine as a latent bioorthogonal electrophilic probe for deubiquitylating enzymes

J. Am. Chem. Soc. 2016

We report the development of two facile selenocysteine-based strategies to generate the DUB probe dehydroalanine (Dha).


Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases

Chembiochem 2016

We demonstrate the utility of the transpeptidase sortase (SrtA) in modifying individual subunits of ubiquitin chains with fluorophores.


Protein Sci. 2016

Here, we compare the structure and function of Ub dimers bearing native and non-native linkages.Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function.


Restricting the ψ Torsion Angle Has Stereoelectronic Consequences on a Scissile Bond: An Electronic Structure Analysis

Biochemistry 2015

We used hybrid density functional theory and natural bond orbital analysis to understand how the stereoelectronic effects of the scissile bond change as a function of φ and ψ torsion angles.Our work reveals important insights into the role of substrate conformation in activating the reactive carbonyl of a scissile bond. These findings have implications for designing potent active site inhibitors based on the concept of transition state analogues.


Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin-protein conjugates

Curr. Opin. Chem. Biol. 2015

We describe recent advances and discuss how chemical methods have been implemented to address the molecular underpinnings of ubiquitin-dependent cellular signaling.


Simultaneous detection of distinct ubiquitin chain topologies by 19F NMR

ACS Chem. Biol. 2014

By exploiting the environmental sensitivity of the 19F nucleus and the conformational diversity found among Ub chains of different linkage types, we simultaneously resolve the 19F NMR signals for mono-Ub and three distinct di-Ub oligomers (K6, K48, and K63) in heterogeneous mixtures. The utility of this approach is demonstrated by the ability to interrogate the selectivity of deubiquitinases with multiple Ub substrates in real time.


Middle-down mass spectrometry enables characterization of branched ubiquitin chains

Biochemistry 2014

We describe a method for the detection of enzymatically derived branched polyUb conjugates in which a single Ub subunit is modified by two Ub molecules at distinct lysine residues. Using a middle-down mass spectrometry approach in which restricted trypsin-mediated digestion is coupled with mass spectrometric analysis, we characterize the polyUb chains produced by bacterial effector E3 ligases NleL (non-Lee-encoded effector ligase from enterohemorrhagic Escherichia coli O157:H7) and IpaH9.8 (from Shigella flexneri).


Chemoenzymatic synthesis of bifunctional polyubiquitin substrates for monitoring ubiquitin chain remodeling

Chembiochem 2014

Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy.


Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product

Biochemistry 2014

We report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively.


Neutron-encoded mass signatures for multiplexed proteome quantification

Nat. Methods 2013

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes.


Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers

Angew. Chem. Int. Ed. Engl. 2012

We report on a method based on thiol-ene chemistry that enables the synthesis and purification of ubiquitin oligomers with ≥4 units. This approach, which employs free-radical polymerization, can be applied towards the synthesis of homogeneous Lys6-linked ubiquitin oligomers currently inaccessible by enzymatic methods. By using these chains, one can study their roles in the ubiquitin proteasome system and the DNA damage response pathway.

JACS Cover 2012.jpg

Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases

J. Am. Chem. Soc. 2012

Chemical methods for modifying proteins enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers.

ACS Chem Biol 2012 Cover.jpg

Unraveling the complexity of ubiquitin signaling

ACS Chem. Biol. 2012

In this Review, we focus on developments in both enzymatic and chemical methods that engender ubiquitin with new chemical and physical properties. Moreover, we highlight how these methods have enabled studies directed toward (i) characterizing enzymes responsible for reversing the ubiquitin modification, (ii) understanding the influence of ubiquitin on protein function and crosstalk with other PTMs, and (iii) uncovering the impact of polyubiquitin chain linkage and length on downstream signaling events.

bottom of page