Mitochondrion

Mutations in mitochondrial DNA (mtDNA) compromise ETC activity and impair oxidative phosphorylation. Since eukaryotic cells contain multiple copies of mtDNA, the resulting phenotype depends on the proportion of mutant mitochondrial genomes (the heteroplasmy level). Using isogenic cell lines carrying similar mtDNA deletions, a linear decline in cellular respiration was observed as mitochondrial DNA heteroplasmy increased. Despite this, cellular redox imbalance did not change until heteroplasmy exceeded 50%. As heteroplasmy increased past 70%, cells also exhibited an integrated stress response (ISR) and impaired translation was observed. These defects were reversed by either addition of asparagine or overexpression of pyruvate carboxylase (PC). The dependence on exogenous asparagine in other respiration-deficient cells was found to correlate inversely with the PC expression level. For example, patient-derived thyroid tumor cells, harboring high heteroplasmy for a Complex I mtDNA mutation and low levels of PC, exhibited asparagine auxotrophy, and L-asparaginase treatment suppressed tumor growth. Together, these findings demonstrate a role for mitochondrial pyruvate carboxylase in cellular asparagine synthesis under conditions of compromised respiratory activity.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

The synthesis of mitochondrial-encoded polypeptides is an essential process, primarily regulated at the posttranscriptional level. In yeast, many regulatory factors have been described as acting in proximity to the mitoribosome to promote efficient translation; however, the precise mechanisms by which these components function remain largely unknown. Here, we expand on findings concerning a previously studied mitoribosome interactor, Mrx9, which is found in large expressosome-like assemblies of mitoribosome clusters. Mrx9 was initially linked to mitochondrial translation and was suggested to be associated with the splicing of COX1 and COB transcripts. Our current data show that Mrx9 is associated with the PHB/m-AAA complex at the polypeptide exit tunnel of the mitoribosome. Overexpression of Mrx9 impairs the proteolytic functions of Yta10 and Yta12 within the prohibitin complex, leading to splicing defects; accumulation of aberrant polypeptides; and a noticeable impairment in the processing of the essential mitoribosomal protein bL32m. These findings support a regulatory role for Mrx9 in the PHB/m-AAA complex by modulating the activities of both Yta10 and Yta12.

Background: Shotgun metagenomic pipelines routinely discard host-derived reads, which can exceed 90% of reads in low-biomass clinical samples. Whether these reads support mitochondrial heteroplasmy detection, and at what allele fraction, has not been established. Methods: Shotgun metagenomic data from 47 upper respiratory tract swabs from healthy adults in Nagpur, India, were analysed. Human Reads were recruited against the rCRS and realigned competitively to GRCh38 to remove nuclear mitochondrial segments (NUMTs). Kraken2 was used as a microbial control. Heteroplasmy was called against a pooled, substitution-specific beta-binomial error model and a leave-one-out panel of normals. The detection limit was evaluated from the substitution spectrum against gnomAD v3.1. Results: Complete mitochondrial genomes were recovered from all samples (mean depth 433X, range 9-3,029X). Competitive realignment relocated 3.22% of recruited reads to nuclear loci; microbial cross-mapping removed 0.045%. Error rates varied 12.8-fold across substitution classes, with T>A being the highest. The 134 calls had a Ti/Tv of 0.43, compared with 3.51 in gnomAD, and T>A accounted for 42.5% of calls. T>A allele fractions never exceeded 4.72%, whereas transitions reached 90.81%. At a 5% threshold, T>A calls disappeared, and Ti/Tv rose to 2.20 before plateauing; 16 calls exceeded this limit. Macrohaplogroups were assigned in 46 of 47 samples (M 22, R 23, N 1). Conclusions: Metagenomic host reads support complete mitochondrial recovery, haplogroup typing, and heteroplasmy detection for allele fractions > 5%. Below this, a T>A-dominated error process persists after NUMT removal that background-fitted models do not capture.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

Mitochondria integrate bioenergetic metabolism, redox control, genome maintenance, and stress signaling across all eukaryotes. Although plant and human mitochondria diverged substantially during evolution, both systems retain systems-level principles for sensing mitochondrial dysfunction and communicating stress signals to the nucleus. Here, we develop an integrative comparative in silico framework to evaluate whether plant mitochondrial stress signaling can provide a useful conceptual model for interpreting human mitochondrial disease vulnerability. Core Arabidopsis thaliana regulators representing alternative respiration, mitochondrial retrograde signaling, translational stress control, and genome surveillance were compared with functionally analogous human regulators involved in integrated stress response (ISR) signaling, mitochondrial DNA maintenance, and mitochondrial disease phenotypes. Domain architecture, protein-protein interaction topology, enrichment profiles, disease-gene associations, and promoter motif architecture were integrated to assess cross-kingdom convergence at the level of stress-response organization rather than direct orthologs. The plant network formed a compact AOX-NAC-centered stress module associated with respiratory flexibility and retrograde signaling, whereas the human network displayed expanded ISR and mtDNA maintenance modules enriched for mitochondrial disease associations. Promoter motif analyses further indicated lineage-specific transcription factor signatures but broadly comparable stress-responsive regulatory logic. Collectively, these results support the concept that plant mitochondrial stress systems represent simplified resilience-oriented architectures that can help generate experimentally testable hypotheses about failure points in human mitochondrial stress responses.

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.

Mitochondrial genomes of angiosperms are unusual due to their large size, low gene content, and high structural variation. In the palm family (Arecaceae), mitochondrial architecture is highly dynamic, characterized by expansive sizes, fragmentation, and extensive recombination activity. This biological complexity presents a significant bioinformatic challenge, especially since only three complete palm mitochondrial genomes are publicly available to date. Here, we present the complete mitochondrial genome sequence for the Amazonian palm Euterpe precatoria, assembled de novo using PacBio HiFi long-read sequencing data. The assembly required extensive manual curation and graph tracing to resolve a complex recombination topology. The final resolved sequence spans 1,268,308 base pairs and is supported by a mean read coverage depth estimated at 746X. Annotation revealed a high degree of structural complexity, including 177 total gene features corresponding to 76 unique genes. This gene set includes 41 unique protein-coding genes (56 features), 32 unique tRNA genes (118 features), and three unique rRNA genes, reflecting the characteristic gene duplication and fragmentation documented in Arecaceae. The complete mitochondrial genome sequence is publicly available and provides a high-quality resource for understanding the mechanisms of plant mitochondrial genome expansion and structural evolution.

Language genes can be tentatively considered as a subset of cognitive genes, although they are often discussed separately. During the evolution of SNVs (single nucleotide variations) in cognition-related genes, do language genes and cognitive genes exhibit significantly different intensities of change at several key evolutionary moments--namely, the inflection points or derivative peak positions of similarity curves drawn from multi-SNV locus bases across samples? In this study, nine distance/similarity metrics (Bray-Curtis, Cosine, Pearson, Spearman, Hamming, Jaccard, Matching, Kulczynski, and Gower) were employed to analyze 413 samples from 11 taxonomic groups, targeting SNV loci in language/cognition-related genes (13,415 effective loci, approximately 400 loci per gene), with pp6 (Homo_sapiens.GRCh38) as the reference. For each method, sample similarities (defined as 1/(1+distance)) were independently sorted in ascending order to generate raw similarity scatterplots. Due to the large sample size and representativeness, the scatter density on the similarity curves was high, and no smoothing was applied. Derivative values were calculated from adjacent similarity differences to identify peaks of evolutionary rate change (top 10 peaks per method). Combined with functional annotations of 33 language/cognition-related genes, we quantified the difference scores and occurrence frequencies of the two gene categories at the peak positions. The results indicate that cognitive-related genes exhibit slightly higher occurrence frequencies in peak windows and higher average difference scores per gene than language genes. Comparative analysis of SNVs at the peak samples and their left-side windows revealed that at positions 381-382, all nine methods shared three intersecting mutation loci, involving language genes (NFXL1, SRGAP2, SRGAP2C); at positions 355-356, there was one intersecting mutation locus, involving a language gene (SRGAP2). This suggests that certain mutations in language genes may have played a distinctive role at critical junctures in the evolution of cognitive abilities.

Background: Macrophages play a central role in systemic iron homeostasis by recycling iron from senescent erythrocytes through the only known cellular iron exporter, ferroportin (Fpn). Previous studies have shown that Fpn localizes to membrane microdomains, or lipid rafts, which are important for its function and regulation by hepcidin. Iron strongly promotes the enrichment of Fpn in lipid rafts. However, the proteomic composition of these domains during iron overload remains largely unexplored. Objective: This study aimed to characterize iron-induced changes in the macrophage lipid raft proteome in order to identify potential functional partners of Fpn and better define the cellular mechanisms involved in the response to iron overload. Methods: Quantitative iTRAQ-based proteomic analysis was performed on detergent-resistant membranes (DRMs) isolated by iodixanol density-gradient ultracentrifugation from J774a.1 macrophages treated with iron-NTA (FeNTA). Key proteomic observations were further validated in both J774a.1 cells and bone marrow-derived macrophages (BMDMs) using western blot, cell-surface biotinylation, and confocal immunofluorescence microscopy. The regulatory role of the transcription factor Nrf2 was also assessed using Nrf2-knockout mouse models. Results: In addition of Fpn, our lipid raft proteome identified 79 proteins significatively upregulated upon iron treatment, including, antioxidant proteins like the peroxiredoxin-1 (Prdx1), the glucose-6-phosphate dehydrogenase X-linked (G6pdx), as well as heme oxygenase-1 (Hmox1). Bioinformatic and functional analyses indicated that this response is associated with nuclear translocation of Nrf2, which is required for the transcriptional induction of Fpn and Hmox1. Whereas Fpn was detected in lipid rafts under basal conditions, Hmox1 was recruited to these domains only after iron or heme treatment. Confocal microscopy and cell-surface biotinylation assays confirmed that Hmox1 and Fpn colocalize at the plasma membrane, particularly within caveolae, specialized invaginated lipid raft domains. Conclusions: Iron or heme overload induces a major remodeling of the macrophage membrane, leading to the formation of specialized lipid raft platforms enriched in antioxidant and iron handling proteins. The coordinated localization of Hmox1 and Fpn at the cell surface suggests a coupled mechanism linking heme catabolism to iron export. This organization may help limit intracellular iron accumulation and protect the plasma membrane from iron-mediated lipid peroxidation.

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

Mitochondria are cellular energy hubs best known for ATP production via oxidative phosphorylation; however, they also serve as biosynthetic centers for phospholipids. Mitochondrial phospholipids are critical for various cellular processes, and their loss underlies myriad mitochondrial diseases. The critical enzymes underlying these biosynthetic cascades are encoded in the nucleus, translated in the cytosol, and imported into mitochondria. Understanding of mechanisms and factors that ensure precise targeting of proteins to mitochondria has been long overlooked but remains critical. Recently, the J-protein/Hsp40 cochaperone Djp1 has emerged as a key player in mitochondrial protein targeting by promoting the transfer of precursors from the endoplasmic reticulum (ER) surface to mitochondria in a pathway termed ER-SURF. Molecular details regarding how Djp1 recognizes clients and more broadly supports mitochondrial function remain unknown. Using biochemical approaches, proteomics, and thin layer chromatography, we demonstrate that Djp1 is a regulator of Phosphatidylserine decarboxylase 1 (Psd1), an inner mitochondrial membrane resident responsible for mitochondrial phosphatidylethanolamine (PE) production. This regulation of Psd1 biogenesis is dependent on its mitochondrial targeting signal and is specific to Djp1 compared to other members of the Hsp40 family or ER targeting factors. Intriguingly, the combined loss of Djp1 and Psd1 results in a synthetic sick phenotype that unexpectedly reflects a role(s) for Djp1 in proper mitochondrial phospholipid metabolism independent of Psd1. Taken together, these findings expand our understanding of Djp1-dependent mitochondrial protein regulation and unveil Djp1 as important for mitochondrial phospholipid metabolism by multiple mechanisms.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Inter-organellar communication is crucial for cellular function. Inside the cell, organelles interact with each other via membrane contact sites (MCSs). These structures mediate the close apposition of two organellar membranes to allow for the exchange of metabolites, ions and lipids. Most of what is known about MCSs comes only from a handful of well-studied metazoans, particularly yeast and mammals. Apicomplexans are parasites that drive human disease throughout the world. Yet, little is known about the makeup or function of their MCSs, leaving a gap in our understanding of how organelles communicate beyond conventional model eukaryotes. Here, we used a proximity biotinylation approach to map the surface proteome of three organelles in the model apicomplexan Toxoplasma gondii: the apicoplast--a non-photosynthetic plastid found only in apicomplexans--its single mitochondrion and the endoplasmic reticulum. By subtracting a cytosolic spatial reference, our high-stringency proteomic analysis uncovered candidate proteins localized simultaneously to multiple organellar surfaces suggesting their role as MCS components. We then validate our approach by characterizing a candidate involved in the association between the apicoplast and the mitochondrion. Overall, our findings provide a valuable approach to identify MCSs in apicomplexans and set the stage to apply our approach to other organelles in these pathogens. HighlightsO_LIGeneration of surface proteomes for the apicoplast, mitochondrion, and ER in Toxoplasma gondii C_LIO_LIMapping of the first endoplasmic reticulum and mitochondrial surface proteomes in T. gondii C_LIO_LIIdentified novel membrane contact site candidate proteins C_LIO_LIValidated a membrane contact site candidate mediating mitochondrion-apicoplast interactions C_LI

This study aims to quantify the genetic similarity of different species (from fish to humans) to the human reference genome (pp6, Homo sapiens.GRCh38) based on the allele presence/absence patterns of 33 language/cognition related gene SNV loci, identify key breakpoints during evolution, and evaluate the enrichment of language and cognition genes at these breakpoints. We designed a similarity calculation method relying on binary features (four columns for A/T/C/G), adopted five difference/distance measures (Sorensen, Rogers, Nei, Reynolds, and Hellinger), and converted them into similarity values (1/(1+distance)). For each method, samples were independently ranked, the first derivative of similarity was computed, and the top 12 peaks were selected as candidate breakpoints. Results show that the similarity curves from the five methods are highly consistent (correlation coefficients >0.9), with major peaks concentrated at positions 355, 363, 381, 382, 390, 400, etc., where the corresponding samples are predominantly ancient hominins and primates. Furthermore, we defined 13 peak groups (starting positions 355-401). For each peak within a group, pairwise SNV differences between the peak apex sample and its immediate left neighbor were compared, and the intersection F_INTERSECTION (shared differential loci) was obtained. For each F_INTERSECTION, we calculated the proportions of language genes and cognition genes. In addition, we computed the differential sets between adjacent groups' F_INTERSECTION to trace the gradual emergence of new loci. In F_INTERSECTION, language genes accounted for an average of 59.5%, and cognition genes for an average of 62.9%. The proportion of language genes reached a peak at position 383 (61.2%), while cognition genes peaked at position 386 (64.9%). High frequency peak samples include c25, c27, and ja2, suggesting that language cognition genes may have undergone independent intensification during Eurasian evolution. Differential analysis between adjacent F_INTERSECTION revealed a stepwise acquisition of new loci from position 355 to 401, with three bursts of newly added loci along the entire evolutionary axis. This study provides a quantitative framework based on similarity curves, offers a novel molecular perspective for understanding the evolution of language and cognitive abilities, and highlights the potential importance of East Asian archaic hominins in the evolution of language cognition genes.

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCA{beta} from Agave tequilana (AtRCA) in Setaria viridis. A second line was created where the endogenous SvRCA{beta} was substituted for AtRCA{beta} through complementation of a null mutant, {triangleup}rcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO2 assimilation at 25{degrees}C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates than AtRCA, however, after 24 hours CO2 assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C4 model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.

Infantile cardiomyopathies are rare, life-threatening disorders for which genetic diagnosis has been accelerated by next-generation sequencing approaches, including gene panel, exome, and genome sequencing. However, determining the functional consequences of identified variants remains a major challenge. Variants in SMYD1, a striated muscle-specific lysine methyltransferase critical for cardiac development and mitochondrial function, have only recently been linked to human cardiomyopathy. Here, we functionally characterize a homozygous SMYD1 variant (c.302A>G; p.Asn101Ser) identified in a patient with severe early-onset cardiomyopathy requiring cardiac transplantation. Structural modeling predicts that the N101S substitution perturbs a highly conserved residue near the cofactor binding pocket within SMYD1s catalytic domain, disrupting local interactions and modestly destabilizing the protein. Consistent with these predictions, in vitro studies demonstrate that the N101S variant impairs mitochondrial respiratory capacity in myocytes. Quantification of SMYD1 protein levels in patient cardiac tissue revealed increased SMYD1 abundance, suggesting that the N101S variant results in functional impairment rather than protein instability and may trigger compensatory upregulation of SMYD1 expression. Together, these findings support a hypomorphic mechanism in which the N101S variant disrupts SMYD1 activity, leading to mitochondrial dysfunction and cardiomyopathy. This study provides mechanistic insight into SMYD1-associated cardiomyopathy and highlights the importance of integrating genetic, structural, and functional analyses to establish the pathogenicity of rare variants. New & NoteworthyThis study provides the first functional characterization of the cardiomyopathy-associated SMYD1 N101S variant identified in a child with severe infantile cardiomyopathy. Structural modeling predicts reduced protein stability, while cellular assays demonstrate impaired mitochondrial respiratory function, supporting a hypomorphic effect. These findings establish a mechanistic link between SMYD1 dysfunction and infantile cardiomyopathy and highlight the importance of integrating genomic and functional approaches in rare cardiovascular disease.

Abstract Optic Atrophy 1 (OPA1), an important inner mitochondrial membrane GTPase, regulates mitochondrial fusion, maintains cristae structure, calcium buffering, cellular bioenergetics, preserves mtDNA and controls apoptosis. Here we examined the role of OPA1 variants in DCM using whole-exome sequencing (WES) of 5 familial and 10 sporadic DCM cases. A rare de novo OPA1 variant, c.563C>T (p.Pro188Leu), was identified in a DCM patient, which is absent in 100 healthy controls as well as in the 1000 Genomes, IndiGenomes and GenomeAsia 100k databases while it showed very low MAF (0.000069) in GnomAD. Structural modelling predicted the variant to be highly deleterious and revealed marked conformational distortion of the mutant protein (RMSD = 3.5 Angstrom). Molecular docking further demonstrated enhanced accessibility of mutant OPA1 to mitochondrial protease OMA1, suggesting increased OPA1 proteolytic processing and a consequent increase in mitochondrial fragmentation. Functional analysis in stable H9C2 cardiomyoblast cells, demonstrated significantly reduced OPA1 protein expression, extensive mitochondrial fragmentation in mutant-OPA1 expressing cells. The mutant protein caused significant reduction in mitochondrial membrane potential, ATP generation, and oxygen consumption rate (OCR), together with elevated cytosolic Calcium and reactive oxygen species (ROS) levels. qRT-PCR analysis further revealed depletion in mtDNA copy number and increased in expression of intrinsic apoptotic markers Caspase3, 9 and Bax/Bcl-2 ratio. The above findings collectively highlighted the significant impact of the OPA1 mutation on mitochondrial dynamics and cellular health, suggesting a significant correlation with the pathogenesis of DCM. Collectively, these findings suggest that OPA1-mediated mitochondrial dysfunction represents a potential therapeutic avenue for the management of DCM.

Telomeres are nucleoprotein structures that protect chromosome ends and are maintained by the Telomerase Reverse Transcriptase (TERT) protein that uses a noncoding Telomerase RNA (TR) as a template. In monkeyflowers, Mimulus lewisii had an ancient TR gene duplication, synthesizing an evolutionarily atypical sequence heterogeneous telomere. How TERT interacts with both TR paralogs during telomere maintenance is unknown and answers can shed novel insights underlying telomere function. Using new genome assemblies we discovered TERT is rapidly evolving in lineages sharing the TR duplication. We investigated the functional consequences arising from the rapid evolution, first by using yeast three-hybrid and testing the physical binding between conspecific and heterospecific TERT-TR combinations. Results showed TERT binds both ancestral (TR1) and derived (TR2) TR paralogs in M. lewisii, but not in species without a functioning TR2. We located the region of TR binding to amino acids near the KRxR motif. We then combined next-generation sequencing with Telomeric Repeat Amplification Protocol and discovered M. lewisii had high telomerase activity. Comparative transcriptomics indicated no strong evidence of expression divergence in telomere maintenance genes for M. lewisii, suggesting rapid evolution shaped TERT protein sequence. In vivo activity of M. lewisii telomerase was investigated by analyzing F1 telomeres generated by crossing M. lewisii and M. verbenaceus, which doesnt have a functioning TR2. Results showed M. verbenaceus chromosome ends in the F1 had converted into M. lewisii telomeres, suggesting dominance of the M. lewisii telomerase. We demonstrate TERT-TR coevolution can have significant consequences on the evolution of plant telomeres. Significance statementTelomeres protect chromosome ends and are maintained by the telomerase complex. We discovered the catalytic component of the telomerase (TERT) was rapidly evolving in monkeyflowers (Mimulus) and studied the molecular consequences. In M. lewisii, TERT evolved lineage-specific amino acids to bind two sequence divergent telomerase RNA paralogs. Telomerase activity assay showed M. lewisii synthesized more telomere repeats compared to its sister species without the TR duplication, and transcriptomics indicated this was not due to a change in telomere maintenance gene expression. Genetic experiments in interspecies hybrids showed M. lewisii telomerase could convert chromosome ends in sister species into M. lewisii-like telomeres suggesting functional dominance. We show rapid evolution of the telomerase can have significant effects on telomere evolution.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.