Search for: All records

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored. 

The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and Mössbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism. 

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD⁺-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.

 

Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform‐specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano‐DESI MSI) for the proteoform‐selective imaging of biological tissues. Nano‐DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof‐of‐concept experiments demonstrate that nano‐DESI MSI combined with on‐tissue top‐down proteomics is ideally suited for the proteoform‐selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.

 

Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform‐specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano‐DESI MSI) for the proteoform‐selective imaging of biological tissues. Nano‐DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof‐of‐concept experiments demonstrate that nano‐DESI MSI combined with on‐tissue top‐down proteomics is ideally suited for the proteoform‐selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.