MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter

The mitochondrial uniporter is a selective Ca²⁺ channel regulated by MICU1, an EF hand‐containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co‐expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology. We report that HEK‐293T cells lacking MICU1 or MICU2 lose a normal threshold for Ca²⁺ intake, extending the known gating function of MICU1 to MICU2. Expression of MICU1 or MICU2 mutants lacking functional Ca²⁺‐binding sites leads to a striking loss of Ca²⁺ uptake, suggesting that MICU1/2 disinhibit the channel in response to a threshold rise in [Ca²⁺]. MICU2's activity and physical association with the pore require the presence of MICU1, though the converse is not true. We conclude that MICU1 and MICU2 are nonredundant and together set the [Ca²⁺] threshold for uniporter activity.     

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA^Met) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA^Met) for both initiation and elongation. A portion of Met-tRNA^Met is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria

Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression data were largely concordant with large-scale surveys of RNA abundance and both measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger "neighborhood" of genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's contribution to human disease.     

Management of adverse events associated with bosutinib treatment of chronic-phase chronic myeloid leukemia: expert panel review

Bosutinib, a BCR-ABL1 tyrosine kinase inhibitor (TKI), has been available for several years as a treatment for chronic-, accelerated-, and blast-phase chronic myeloid leukemia (CML), for patients with resistance or intolerance to prior therapy. In 2017, the BFORE trial demonstrated efficacy of bosutinib as first-line treatment in adult patients with newly diagnosed chronic-phase chronic myeloid leukemia (CP-CML). The most common adverse events (AEs) of any grade in bosutinib-treated patients in BFORE were diarrhea, nausea, thrombocytopenia, increased alanine aminotransferase, and increased aspartate aminotransferase, consistent with the most commonly reported AEs in earlier studies. To balance the efficacy and tolerability of treatment to optimize patient adherence with medications, treating physicians commonly use various strategies such as initiating treatment at a lower dose, dose reduction, or dose interruption, depending on the type and severity of the AEs and the clinical setting. In light of the recent data from first-line treatment, an expert panel of hematologists reviewed management strategies for the use of bosutinib in treatment of CP-CML and made the recommendations reported here. Although the panel focused on first-line treatment, the principles can be for the most part extended to bosutinib use in later lines of treatment. Recommendations include advice regarding prophylaxis and management for diarrhea. The panel also considered optimum timing for referral to a specialist for specific AEs. Across the commonly occurring AEs, the panel highlighted the importance of education and communication with patients about anticipated AEs.