Dysregulated dopamine (DA) signaling and redox homeostasis contributes to multiple neuropsychiatric and neurodegenerative disorders. Polymorphisms that influence the activity of catechol-O-methyltransferase (COMT), an enzyme critical for degrading DA in the dorsolateral prefrontal cortex, have been implicated in behavioral and neuropsychiatric alterations associated with schizophrenia (SCZ). Adverse neuropsychiatric effects have also been reported in Parkinson's disease (PD) patients administered COMT inhibitors in combination with other DA-enhancing therapies. COMT exists as two isoforms: a soluble short isoform (S-COMT) and a membrane-bound long isoform (MB-COMT). These variants differ in their N-terminal domains, with MB-COMT being the predominant brain isoform. Here, unbiased proteomic and biochemical analyses show that genetic loss of MB-COMT disrupts DA signaling and perturbs pathways governing synaptic and mitochondrial function, iron and copper homeostasis, and redox balance. Limited proteolysis mass spectrometry (LiP-MS) further revealed that MB-COMT deficiency triggers widespread protein structural alterations, a molecular event commonly occurring in neurodegenerative conditions but not as well studied in neuropsychiatric diseases. Our results show that MB-COMT is a molecular hub that connects multiple cellular pathways whose differential dysregulation underlies the pathophysiology of complex neuropsychiatric diseases such as SCZ. Thus, MB-COMT is identified as a key regulator of brain DA biology, loss of which activates cellular stress response pathways, revealing potential targets for therapeutic intervention.
S. Tripathi, Suwarna Chakraborty, Neil B. Wood et al.· Proceedings of the National...· 0 citations
Cross-linking mass spectrometry (XL-MS) has advanced as a powerful approach to map protein-protein interactions in cells. With recent advances in cross-linkers, instrumentation, and software, interactions can be observed in a proteome-wide fashion at residue-level resolution. In most XL-MS experiments, a chemical cross-linker possessing two electrophiles is added to a biological sample and records spatial information by forming covalent bonds between two proximal nucleophilic residues. On the other hand, photo-cross-linking amino acids that can be incorporated into proteins via ribosomal synthesis possess the capacity to rapidly capture transient protein-protein interactions under physiological conditions without introducing exogenous reactive species. Diazirine-based photoamino acids have been widely used for this purpose due to their small size and broad reactivity. Recent studies have shown that diazirines can form MS-cleavable linkages when they react with acidic residues. However, confident interpretation of the resulting spectra and localization of cross-linking sites remain challenging, limiting broader application of diazirine-based approaches in proteome-wide cross-linking analyses. Here, we address this limitation by leveraging DizPK, a diazirine-containing lysine analog with a dedicated MS-cleavable urea functionality. We demonstrate proteome-wide incorporation of DizPK in Escherichia coli via stochastic orthogonal recoding of translation and subsequent identification of photo-cross-linked peptides to map protein-protein interactions under physiological conditions. We find that the resulting approach can provide high-resolution structural information associated with transient biological processes.
Piyoosh Sharma, Divya Yadav, Anneliese M. Faustino et al.· Journal of Proteome Research· 0 citations
Pichia pastoris is a widely used host for recombinant protein production because it combines the advantages of microbial cultivation with eukaryotic protein folding and secretion. However, secretion efficiency is often limited by the folding capacity of the endoplasmic reticulum (ER), where recombinant proteins must be translocated, folded, and processed prior to export. When ER folding capacity is exceeded, proteins may be retained, degraded, or secreted in non-native conformations, reducing both yield and product quality. Chaperone engineering and codon optimization represent two promising strategies to address these limitations. Here, we generated stable Pichia strains expressing four model secreted proteins (human serum albumin, interleukin-2, thaumatin-I, and thaumatin-II) using either conventional codon optimization or Epi-MAX codon engineering, which adapts transgene codon usage to stress-responsive translational programs. We also engineered strains containing an additional chromosomal copy of either the ER Hsp70 chaperone Kar2 or protein disulfide isomerase (Pdi1). To assess protein quality, we applied limited proteolysis mass spectrometry (LiP-MS), a structural proteomics approach that can detect subtle conformational differences to secreted proteins. Increased Pdi1 levels improved secretion of all four proteins tested, whereas Kar2 overexpression generally reduced yield. For thaumatin-II, Pdi1 enhanced secretion but promoted release of a non-native conformation, which we could correct through codon engineering. Together, these results demonstrate that maximizing recombinant protein production requires optimization of both yield and structural quality and establish complementary strategies for improving secreted protein expression in Pichia.
