Browsing by Author "Zhou, Pei"

Addition and removal of phosphate is an important post-translational modification involved in cellular signaling. The enzymes responsible for removing this phosphorylation mark, called phosphatases, play a vital role in the cellular decision making processes. In this work we discuss two discoveries, a novel enzyme for a known signaling function involving control of transcription and a novel target for an important cellular stress response enzyme.

In the first project we sought to determine a novel enzyme responsible for dephosphorylating the C-terminal domain of RNA polymerase II. This domain serves as a vital signaling platform for transcription of mammalian genes, with the ability to recruit cofactors that bind to specific patterns of phosphorylation throughout its repeating amino acid sequence. Using a functional assay for phosphatase activity at the Thr4 position we biochemically isolated the unknown enzyme and identified it as PP1 and validated its function in vitro and in vivo.

The second phosphatase studied in this dissertation is MESH1—a mammalian ortholog of the bacterial stringent response protein SpoT that dephosphorylates ppGpp. Because ppGpp is absent in mammalian cells MESH1 lacks a viable target. We established NADPH as a substrate of MESH1 biochemically and corroborated these results by determining the substrate bound structure. Our results reveal a novel regulatory role of MESH1 in a pathway that resembles the bacterial stringent response.

The outer-leaflet of the outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS), which is attached to the membrane via a hexa-acylated saccharolipid called lipid A. The fourth step of lipid A biosynthesis involves the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN to form lipid X; this step is carried out by LpxH in E. coli and the majority of Gamma- and Beta-Proteobacteria. LpxH has been previously characterized, however sample impurity and non-optimized assay conditions hindered meaningful conclusions. The enzyme was suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes, however the extent of biochemical data fails to demonstrate a significant level of metal activation in LpxH assays. We report cloning, purification, and detailed enzymatic characterization with a highly purified sample of H. influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of activity in the presence of Mn2+. Furthermore, EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Overall, through optimized purification and assay methods, our work unambiguously establishes LpxH as a membrane-associating CLP containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.

Several species of Gram-negative bacteria lack LpxH orthologs, yet retain other lipid A biosynthetic enzymes and still produce lipid A. An unrelated protein, LpxI, is responsible for UDP-DAGn hydrolysis is several such organisms. Interestingly, some bacteria, such as the human pathogen Chlamydia trachomatis, have neither LpxH nor LpxI orthologs, suggesting the presence of a third UDP-DAGn hydrolase. Through implantation of a novel complementation screen that used a C. trachmatis genomic library and a conditional-lethal lpxH mutant E. coli strain, we were able to identify an open reading frame encoding an new enzyme capable of lipid X production. Due to its ability to complement UDP-DAGn hydrolase function in vivo and catalyze the formation of lipid X in vitro, we have designated the enzyme LpxG. Further biochemical analysis with purified LpxG revealed it facilitates hydrolysis through attack on the alpha phosphate of its substrate and is activated by Mn2+ in vitro. LpxG is in the same CLP superfamily as LpxH, however it shows very little homology to LpxH or LpxI. Identification of LpxG improves our understanding of the lipid A biosynthetic pathway in C. trachomatis. More broadly, as limited genetic tools are available for the study of the prevalent pathogen, it provides an advantageous method for the functional screening of other C. trachomatis genes.

Epilepsy is a syndrome that affects about 65 million people around the world. About 150,000 new cases of epilepsy are reported in the United States every year. Temporal lobe epilepsy (TLE) is the most common form of human epilepsy. It is a chronic neurological disorder characterized by recurrent seizures that are devastating due to a lack of effective treatment. TLE is resistant to anticonvulsants and one-third of patients diagnosed with TLE are refractory to medication.

Excessive activation of tropomyosin receptor kinase B (TrkB) promotes TLE. The importance of phospholipase Cγ1 (PLCγ1) as a major downstream signaling effector of TrkB was first identified by the McNamara lab at Duke. Thus, selective inhibition of the PLCγ1-TrkB interaction constitutes a promising avenue for new drugs.

As a proof-of-concept, the McNamara lab engineered a novel 14-mer peptide pY816 that effectively inhibits epilepsy and prevents anxiety-like behavior induced by continuous seizure activity (status epilepticus), in a dose- and time-dependent manner. Despite their promising therapeutic effectiveness, the molecular details of the engagement of these inhibitors with PLCγ1 have remained elusive.

In this study, we propose x-ray crystallography and solution NMR studies to elucidate the binding mode of the peptide to PLCγ1 tandem SH2 domains. This will ultimately facilitate the development of novel therapeutics targeting the TrkB-PLCγ1 interaction.

LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in E. coli, is an essential enzyme for virtually all Gram-negative bacteria and one of the most promising novel antibiotic targets for the treatment of multidrug-resistant Gram-negative infections. Here, we report the characterization of two novel LpxC inhibitors that have apparent binding affinities for E. coli LpxC in the picomolar range. Furthermore, these compounds display broad spectrum activity against a plethora of Gram-negative pathogens.

In anticipation for the advancement of LpxC inhibitors in clinical trials, we undertook studies to probe potential bacterial resistance mechanisms to these compounds. In this study, we report a two-step isolation of spontaneously resistant E. coli mutants that have > 200-fold resistance to LpxC inhibitors. These mutants have two chromosomal point mutations that account for resistance additively and independently: one in fabZ, a dehydrase in fatty acid biosynthesis, and the other in thrS, the Thr-tRNA ligase.

For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Most unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain balance between phospholipid and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, providing the first example that reduced protein biosynthesis confers a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal an impressive compensatory ability of bacteria to overcome inhibition of lipid A biosynthesis by rebalancing cellular homeostasis, a unique mechanism of antibiotic resistance.

Heat Shock transcription Factor 1 (HSF1) has long been recognized as the master regulator and signal integrator in the eukaryotic proteotoxic stress response. Revealed by recent discoveries in cancer, the functions of HSF1 have extended far beyond its canonical role in protein folding, further encompassing critical functions in anti-apoptosis, invasion and metastasis, energy metabolism, DNA damage repair, and evasion of host immune surveillance. Meanwhile, both our understanding of the molecular basis of HSF1 regulation as well as available biochemical tools to investigate such details are lacking. Based on an in vitro ligand binding approach, the studies presented in this thesis were dedicated to the identification, validation, and characterization of a direct, first-in-class, small-molecule HSF1 inhibitor. The pharmacological inhibition of HSF1 occurs through small-molecule stimulation of nuclear, but not cytoplasmic HSF1 degradation, which attenuated prostate cancer cell proliferation, inhibited the HSF1 cancer gene signature and arrested tumor progression in multiple therapy-resistant animal models of prostate cancer. The identification of a direct small-molecule HSF1 inhibitor provides a unique pharmacological tool for future HSF1 research and serves as a significant proof-of-concept for pharmacologically targeting HSF1 for anti-cancer treatment approaches.

Molybdenum cofactor (Moco) is a ubiquitous cofactor essential for lives of most organisms. In humans, Moco is essential for healthy development of brain, and inability to produce Moco causes a fatal Moco deficiency (MoCD) disease. Moco biosynthesis is initiated by a conserved and complicated conversion from GTP to cyclic pyranopterin monophosphate (cPMP). In bacteria, this transformation is catalyzed by two enzymes, MoaA and MoaC. Of these two enzymes, MoaA attracts research interests as it is one of the founding members of the radical S-adenosyl-L-methionine (SAM) superfamily, and its human homolog, MOCS1A, harbors over 50% of MoCD-causing mutations identified in patients. MoaA catalyzes an unprecedented and chemically challenging 3’,8-cyclization of GTP into 3’,8-cyclo-7,8-dihydro-GTP (3’,8-cH2GTP) using an active site containing two 4Fe-4S clusters, three conserved Arg residues near the GTP binding site (R17, 266, 268) and two strictly conserved Gly residues on a disordered C-terminal tail (G339, 340; GG-motif). These amino acid residues were reported to be mutated in human MoCD disease patients. However, the details of catalytic mechanism of MoaA or the mechanism by which these mutations inactivate MoaA remained elusive.In this dissertation, I aimed to elucidate the catalytic mechanism of MoaA and the catalytic roles of amino acid residues whose mutations in human cause the MoCD disease. In Chapter 2, I describe enzymological characterization of the MoaA-catalyzed reaction. In this study, I found a shunt pathway that accumulates 5’-deoxyadenos-4’-yl radical and yields (4’S)-5’-deoxyadenosine. Using this shunt pathway as a reference, I was able to determine the rate constant for the MoaA-catalyzed C3’-C8 cyclization and revealed that MoaA accelerates the rate of radical-mediated C3’-C8 bond formation of GTP by 6 ~ 9 orders of magnitude using R17 to stabilize the transition state. In Chapter 3, I describe characterization of the redox function of 4Fe-4S clusters using a combination of protein film voltammography (PFV), X-band and Q-band EPR, and DFT calculation. Based on the results from these studies, I proposed a proton-coupled electron transfer (PCET) mechanism for the radical quenching step, where the auxiliary 4Fe-4S cluster acts as an electron donor and R17 acts as a proton donor. In Chapter 4, I describe structural and functional characterization of a 11mer peptide that mimics the C-terminal tail and rescues the activity of GG-motif mutants. The structure of the peptide in complex with MoaA was solved by nuclear magnetic resonance (NMR), which suggests that the GG-motif inserts into the active site and likely interacts with active site Arg residues. In Chapter 5, I describe characterization of the MoaA catalytic mechanisms using GTP analogs as mechanistic probes. Overall, this work provides new insights into the mechanisms by which MoaA controls the reactivity of radical intermediates and quenches the radical intermediate at the specific timing. The results have significant implications in understanding the catalytic mechanisms of radical SAM enzymes in general. It also contributes to our enzymological understanding of molecular causes of MoCD and will form foundations for development of novel therapeutics for MoCD.