Metabolic Screening of Cytotoxic T-cell Effector Function Reveals the Role of CRAC Channels in Regulating Lethal Hit Delivery.

Cytotoxic T lymphocytes (CTL) mediate cytotoxicity toward tumor cells by multistep cell-cell interactions. However, the tumor microenvironment can metabolically perturb local CTL effector function. CTL activity is typically studied in two-dimensional (2D) liquid coculture, which is limited in recapitulating the mechanisms and efficacy of the multistep CTL effector response.

Redox Homeostasis and Mitochondrial Dynamics.

Within living cells, mitochondria are considered relevant sources of reactive oxygen species (ROS) and are exposed to reactive nitrogen species (RNS). During the last decade, accumulating evidence suggests that mitochondrial (dys)function, ROS/RNS levels, and aberrations in mitochondrial morphology are interconnected, albeit in a cell- and context-dependent manner. Here it is hypothesized that ROS and RNS are involved in the short-term regulation of mitochondrial morphology and function via non-transcriptional pathways.

Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis.

Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse.

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits.

Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly.

Mammalian mitochondrial complex I: biogenesis, regulation, and reactive oxygen species generation.

Virtually every mammalian cell contains mitochondria. These double-membrane organelles continuously change shape and position and contain the complete metabolic machinery for the oxidative conversion of pyruvate, fatty acids, and amino acids into ATP. Mitochondria are crucially involved in cellular Ca2+ and redox homeostasis and apoptosis induction.

Baculovirus complementation restores a novel NDUFAF2 mutation causing complex I deficiency.

Mitochondrial complex I deficiency is the most common defect of the OXPHOS system. We report a patient from consanguineous parents with a complex I deficiency expressed in skin fibroblasts. Homozygosity mapping revealed several homozygous regions with candidate genes, including the gene encoding an assembly factor for complex I, NDUFAF2.

Chapter 7 Tracing human mitochondrial complex I assembly by use of GFP-tagged subunits.

Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation.

Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells.

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved.

NDUFA2 complex I mutation leads to Leigh disease.

Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5.

Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly.

Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns.