Co-fractionation-mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain.

Human mitochondrial (mt) protein assemblies are vital for neuronal and brain function, and their alteration contributes to many human disorders, e.g., neurodegenerative diseases resulting from abnormal protein-protein interactions (PPIs). Knowledge of the composition of mt protein complexes is, however, still limited. Affinity purification mass spectrometry (MS) and proximity-dependent biotinylation MS have defined protein partners of some mt proteins, but are too technically challenging and laborious to be practical for analyzing large numbers of samples at the proteome level, e.g., for the study of neuronal or brain-specific mt assemblies, as well as altered mtPPIs on a proteome-wide scale for a disease of interest in brain regions, disease tissues or neurons derived from patients. To address this challenge, we adapted a co-fractionation-MS platform to survey native mt assemblies in adult mouse brain and in human NTERA-2 embryonal carcinoma stem cells or differentiated neuronal-like cells. The workflow consists of orthogonal separations of mt extracts isolated from chemically cross-linked samples to stabilize PPIs, data-dependent acquisition MS to identify co-eluted mt protein profiles from collected fractions and a computational scoring pipeline to predict mtPPIs, followed by network partitioning to define complexes linked to mt functions as well as those essential for neuronal and brain physiological homeostasis. We developed an R/CRAN software package, Macromolecular Assemblies from Co-elution Profiles for automated scoring of co-fractionation-MS data to define complexes from mtPPI networks. Presently, the co-fractionation-MS procedure takes 1.5-3.5 d of proteomic sample preparation, 31 d of MS data acquisition and 8.5 d of data analyses to produce meaningful biological insights.

Sanitation enzymes: Exquisite surveillance of the noncanonical nucleotide pool to safeguard the genetic blueprint.

Reactive oxygen species (ROS) are common products of normal cellular metabolism, but their elevated levels can result in nucleotide modifications. These modified or noncanonical nucleotides often integrate into nascent DNA during replication, causing lesions that trigger DNA repair mechanisms such as the mismatch repair machinery and base excision repair. Four superfamilies of sanitization enzymes can effectively hydrolyze noncanonical nucleotides from the precursor pool and eliminate their unintended incorporation into DNA. Notably, we focus on the representative MTH1 NUDIX hydrolase, whose enzymatic activity is ostensibly nonessential under normal physiological conditions. Yet, the sanitization attributes of MTH1 are more prevalent when ROS levels are abnormally high in cancer cells, rendering MTH1 an interesting target for developing anticancer treatments. We discuss multiple MTH1 inhibitory strategies that have emerged in recent years, and the potential of NUDIX hydrolases as plausible targets for the development of anticancer therapeutics.

Mitochondria under the spotlight: On the implications of mitochondrial dysfunction and its connectivity to neuropsychiatric disorders.

Neuropsychiatric disorders (NPDs) such as bipolar disorder (BD), schizophrenia (SZ) and mood disorder (MD) are hard to manage due to overlapping symptoms and lack of biomarkers. Risk alleles of BD/SZ/MD are emerging, with evidence suggesting mitochondrial (mt) dysfunction as a critical factor for disease onset and progression. Mood stabilizing treatments for these disorders are scarce, revealing the need for biomarker discovery and artificial intelligence approaches to design synthetically accessible novel therapeutics. Here, we show mt involvement in NPDs by associating 245 mt proteins to BD/SZ/MD, with 7 common players in these disease categories. Analysis of over 650 publications suggests that 245 NPD-linked mt proteins are associated with 800 other mt proteins, with mt impairment likely to rewire these interactions. High dosage of mood stabilizers is known to alleviate manic episodes, but which compounds target mt pathways is another gap in the field that we address through mood stabilizer-gene interaction analysis of 37 prescriptions and over-the-counter psychotropic treatments, which we have refined to 15 mood-stabilizing agents. We show 26 of the 245 NPD-linked mt proteins are uniquely or commonly targeted by one or more of these mood stabilizers. Further, induced pluripotent stem cell-derived patient neurons and three-dimensional human brain organoids as reliable BD/SZ/MD models are outlined, along with multiomics methods and machine learning-based decision making tools for biomarker discovery, which remains a bottleneck for precision psychiatry medicine.

Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.