Overexpression of the plastidial pseudo-protease AtFtsHi3 enhances drought tolerance while sustaining plant growth.

With climate change, droughts are expected to be more frequent and severe, severely impacting plant biomass and quality. Here, we show that overexpressing the Arabidopsis gene AtFtsHi3 (FtsHi3OE) enhances drought-tolerant phenotypes without compromising plant growth. AtFtsHi3 encodes a chloroplast envelope pseudo-protease; knock-down mutants (ftshi3-1) are found to be drought tolerant but exhibit stunted growth. Altered AtFtsHi3 expression therefore leads to drought tolerance, while only diminished expression of this gene leads to growth retardation. To understand the underlying mechanisms of the enhanced drought tolerance, we compared the proteomes of ftshi3-1 and pFtsHi3-FtsHi3OE (pFtsHi3-OE) to wild-type plants under well-watered and drought conditions. Drought-related processes like osmotic stress, water transport, and abscisic acid response were enriched in pFtsHi3-OE and ftshi3-1 mutants following their enhanced drought response compared to wild-type. The knock-down mutant ftshi3-1 showed an increased abundance of HSP90, HSP93, and TIC110 proteins, hinting at a potential downstream role of AtFtsHi3 in chloroplast pre-protein import. Mathematical modeling was performed to understand how variation in the transcript abundance of AtFtsHi3 can, on the one hand, lead to drought tolerance in both overexpression and knock-down lines, yet, on the other hand, affect plant growth so differently. The results led us to hypothesize that AtFtsHi3 may form complexes with at least two other protease subunits, either as homo- or heteromeric structures. Enriched amounts of AtFtsH7/9, AtFtsH11, AtFtsH12, and AtFtsHi4 in ftshi3-1 suggest a possible compensation mechanism for these proteases in the hexamer.

Multi-OMICS study of a CHCHD10 variant causing ALS demonstrates metabolic rewiring and activation of endoplasmic reticulum and mitochondrial unfolded protein responses.

Mutations in CHCHD10, coding for a mitochondrial intermembrane space protein, are a rare cause of autosomal dominant amyotrophic lateral sclerosis. Mutation-specific toxic gain of function or haploinsufficiency models have been proposed to explain pathogenicity. To decipher the metabolic dysfunction associated with the haploinsufficient p.R15L variant, we integrated transcriptomic, metabolomic and proteomic data sets in patient cells subjected to an energetic stress that forces the cells to rely on oxidative phosphorylation for ATP production. Patient cells had a complex I deficiency that resulted in an increased NADH/NAD+ ratio, diminished TCA cycle activity, a reorganization of one carbon metabolism and an increased AMP/ATP ratio leading to phosphorylation of AMPK and inhibition of mTORC1. These metabolic changes activated the unfolded protein response (UPR) in the ER through the IRE1/XBP1 pathway, upregulating downstream targets including ATF3, ATF4, CHOP and EGLN3, and two cytokine markers of mitochondrial disease, GDF15 and FGF21. Activation of the mitochondrial UPR was mediated through an upregulation of the transcription factors ATF4 and ATF5, leading to increased expression of mitochondrial proteases and heat shock proteins. There was a striking transcriptional up regulation of at least seven dual specific phosphatases, associated with an almost complete dephosphorylation of JNK isoforms, suggesting a concerted deactivation of MAP kinase pathways. This study demonstrates that loss of CHCHD10 function elicits an energy deficit that activates unique responses to nutrient stress in both the mitochondria and ER, which may contribute to the selective vulnerability of motor neurons.

Loss of CHCHD10-CHCHD2 complexes required for respiration underlies the pathogenicity of a CHCHD10 mutation in ALS.

Coiled-helix coiled-helix domain containing protein 10 (CHCHD10) and its paralogue CHCHD2 belong to a family of twin CX9C motif proteins, most of which localize to the intermembrane space of mitochondria. Dominant mutations in CHCHD10 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia, and mutations in CHCHD2 have been associated with Parkinson's disease, but the function of these proteins remains unknown. Here we show that the p.R15L CHCHD10 variant in ALS patient fibroblasts destabilizes the protein, leading to a defect in the assembly of Complex I, impaired cellular respiration, mitochondrial hyperfusion, an increase in the steady-state level of CHCHD2, and a severe proliferation defect on galactose, a substrate that forces cells to synthesize virtually all of their ATP aerobically. CHCHD10 and CHCHD2 appeared together in distinct foci by immunofluorescence analysis and could be quantitatively immunoprecipitated with antibodies against either protein. Blue native polyacrylamide gel electrophoresis analyses showed that both proteins migrated in a high molecular weight complex (220 kDa) in control cells, which was, however, absent in patient cells. CHCHD10 and CHCHD2 levels increased markedly in control cells in galactose medium, a response that was dampened in patient cells, and a new complex (40 kDa) appeared in both control and patient cells cultured in galactose. Re-entry of patient cells into the cell cycle, which occurred after prolonged culture in galactose, was associated with a marked increase in Complex I, and restoration of the oxygen consumption defect. Our results indicate that CHCHD10-CHCHD2 complexes are necessary for efficient mitochondrial respiration, and support a role for mitochondrial dysfunction in some patients with ALS.

Matchmaking facilitates the diagnosis of an autosomal-recessive mitochondrial disease caused by biallelic mutation of the tRNA isopentenyltransferase (TRIT1) gene.

Deleterious variants in the same gene present in two or more families with overlapping clinical features provide convincing evidence of a disease-gene association; this can be a challenge in the study of ultrarare diseases. To facilitate the identification of additional families, several groups have created "matching" platforms. We describe four individuals from three unrelated families "matched" by GeneMatcher and MatchMakerExchange. Individuals had microcephaly, developmental delay, epilepsy, and recessive mutations in TRIT1. A single homozygous mutation in TRIT1 associated with similar features had previously been reported in one family. The identification of these individuals provides additional evidence to support TRIT1 as the disease-causing gene and interprets the variants as "pathogenic." TRIT1 functions to modify mitochondrial tRNAs and is necessary for protein translation. We show that dysfunctional TRIT1 results in decreased levels of select mitochondrial proteins. Our findings confirm the TRIT1 disease association and advance the phenotypic and molecular understanding of this disorder.

Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I.

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN, and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. Binuclear Fe-S cluster N1a is not part of this long chain but is located within electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes, while the amount of the complex was only slightly diminished. N1a could not be detected by electron paramagnetic resonance spectroscopy, and unexpectedly, the content of binuclear cluster N1b located on a neighboring subunit was significantly decreased. Because of the lack of N1a and the partial loss of N1b, the variants did not survive detergent extraction from the mutant membranes. Only the C97AE variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. We discuss whether N1a is involved in the physiological electron transfer reaction.

State-of-the-art vitamin D assays: a comparison of automated immunoassays with liquid chromatography-tandem mass spectrometry methods.

BACKGROUND: Vitamin D testing is increasing worldwide. Recently several diagnostic manufacturers including Abbott and Siemens have launched automated 25-hydroxy vitamin D (25OH-D) immunoassays. Furthermore, preexisting assays from DiaSorin and Roche have recently been modified. We compared the performance of 5 automated immunoassays, an RIA and 2 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. METHODS: Aliquots of 170 randomly selected patient samples were prepared and 25OH-D was measured by 2 LC-MS/MS methods, an RIA (DiaSorin), and automated immunoassays from Abbott (Architect), DiaSorin (LIAISON), IDS (ISYS), Roche (E170, monoclonal 25OH-D(3) assay), and Siemens (Centaur). Within-run and between-run imprecision were evaluated by measurement of 5 replicates of 2 serum pools on 5 consecutive days. RESULTS: The LC-MS/MS methods agreed, with a concordance correlation coefficient (CCC) of 0.99 and bias of 0.56 µg/L (1.4 nmol/L). The RIA assay showed a performance comparable to LC-MS/MS, with a CCC of 0.97 and a mean bias of 1.1 µg/L (2.7 nmo/L). All immunoassays measured total 25OH-D (including D(3) and D(2)), with the exception of the Roche assay (D(3) only). Among the immunoassays detecting total 25OH-D, the CCCs varied between 0.85 (Abbott) to 0.95 (LIAISON). The mean bias ranged between 0.2 µg/L (0.5 nmol/L) (LIAISON) and 4.56 µg/L (11.4 nmol/L) (Abbott). The Roche 25OH-D(3) assay demonstrated small mean bias [-2.7 µg/L (-6.7 nmol/L)] [-2.7 µg/L (-6.7 nmol/L)] but a low CCC of just 0.66. Most assays demonstrated good intra- and interassay precision, with CV <10%. CONCLUSIONS: Automated immunoassays demonstrated variable performance and not all tests met our minimum performance goals. It is important that laboratories be aware of the limitations of their assay.

Serum 25-hydroxyvitamin D: a predictor of macrovascular and microvascular complications in patients with type 2 diabetes.

OBJECTIVE: People with diabetes frequently develop vascular disease. We investigated the relationship between blood 25-hydroxyvitamin D (25OH-D) concentration and vascular disease risk in type 2 diabetes. RESEARCH DESIGN AND METHODS: The relationships between blood 25OH-D concentration at baseline and the incidence of macrovascular (including myocardial infarction and stroke) and microvascular (retinopathy, nephropathy, neuropathy, and amputation) disease were analyzed with Cox proportional hazards models and logistic regression in an observational study of patients in the 5-year Fenofibrate Intervention and Event Lowering in Diabetes trial. RESULTS: A total of 50% of the patients had low vitamin D concentrations, as indicated by median blood 25OH-D concentration of 49 nmol/L. These patients with a blood 25OH-D concentration <50 nmol/L had a higher cumulative incidence of macrovascular and microvascular events than those with levels ≥50 nmol/L. Multivariate analysis, stratified by treatment and adjusted for relevant confounders, identified blood 25OH-D concentration as an independent predictor of macrovascular events. A 50 nmol/L difference in blood 25OH-D concentration was associated with a 23% (P = 0.007) change in risk of macrovascular complications during the study, and further adjustments for seasonality, hs-CRP, and physical activity level had little impact. The unadjusted risk of microvascular complications was 18% (P = 0.006) higher during the study, though the excess risk declined to 11-14% and lost significance with adjustment for HbA1c, seasonality, or physical activity. CONCLUSIONS: Low blood 25OH-D concentrations are associated with an increased risk of macrovascular and microvascular disease events in type 2 diabetes. However, a causal link remains to be demonstrated.