Dataset Information

Mitochondrial responses during hyperammonemia

ABSTRACT: Ammonia is a cytotoxic metabolite with pleotropic molecular and metabolic effects in non-hepatic tissue/cells targeting mitochondrial oxidative function that may contribute to post-mitotic senescence. Global molecular responses were determined by analysis of unbiased data followed by experimental validation of critical functional consequences in hyperammonemic differentiated myotubes and skeletal muscle from the portacaval anastomosis (PCA) rat. Integrating whole cell transcriptome with whole cell and mitochondrial proteome from hyperammonemic myotubes identified oxidative dysfunction and senescence pathways as being the most enriched with differentially expressed genes/proteins. Hyperammonemia and ammonia-lowering result in distinct clusters of molecular responses. Functional and metabolic studies in hyperammonemic myotubes showed that defects in electron transport chain (ETC) complexes I and III, loss of supercomplex assembly, decreased ATP synthesis, increased free radical generation with oxidative modification of proteins/lipids and increased expression of senescence associated molecular phenotype (SAMP) that were partially reversed by ammonia lowering. Studies in muscle from PCA rats established physiological relevance of our cellular studies. Dysregulated ammonia metabolism causes mitochondrial dysfunction by transcriptional and translational perturbations in multiple pathways that result in reversible senescence.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat) Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle, Skeletal Muscle Tissue

SUBMITTER: Ling Li

LAB HEAD: Srinivasan Dasarathy

PROVIDER: PXD026955 | Pride | 2022-02-17

REPOSITORIES: Pride

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Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes.

Welch Nicole N Singh Shashi Shekhar SS Kumar Avinash A Dhruba Saugato Rahman SR Mishra Saurabh S Sekar Jinendiran J Bellar Annette A Attaway Amy H AH Chelluboyina Aruna A Willard Belinda B BB Li Ling L Huo Zhiguang Z Karnik Sadashiva S SS Esser Karyn K Longworth Michelle S MS Shah Yatrik M YM Davuluri Gangarao G Pal Ranadip R Dasarathy Srinivasan S

The Journal of biological chemistry 20210731 3

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics ...[more]

PMID: 34343564

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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

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	Cell-24h-AmAc-WD1.raw	Raw
	Cell-24h-AmAc-WD2.raw	Raw
	Cell-24h-AmAc-WD3.raw	Raw
	Cell-24h-AmAc1.raw	Raw
	Cell-24h-AmAc2.raw	Raw