Abstract

   The plateau adaptability and stress resistance of yaks are widely known
   based on their capacity to survive under severe habitat conditions.
   However, a few studies on brain mitochondria have characterized these
   adaptations at the protein level. We identified and quantified the
   brain mitochondrial proteins using isobaric tags for relative and
   absolute quantification (iTRAQ) and Proteomics. Western blotting was
   used to verify changes in the expression of target proteins. A total of
   57 differentially abundant proteins (DAPs) were identified in the yak
   brain tissue. Gene Ontology (GO) analysis showed molecular functions of
   these DAPs including downregulated oxidoreductase activity but
   upregulated coenzyme binding. Significantly enriched biological
   processes were oxidation–reduction process (downregulated) and small
   molecule metabolic processes (upregulated). STRING protein interaction
   analysis indicated a complex interaction between dehydrogenase,
   transaminase, and ATP synthetase families. Reactome pathway analysis
   highlighted that the majority of DAPs participated in aerobic metabolic
   pathways such as metabolism, citric acid cycle, and respiratory
   electron transport. Immunoblotting confirmed that changes in FKBP4 and
   ATPAF2 expression were consistent with the results of mass
   spectrometry. We performed a high-throughput screening to identify DAPs
   in brain mitochondria between yak and cattle, which could explain the
   plateau adaptability of yaks.

   Keywords: yak (Bos grunniens), mitochondria, proteomics, plateau
   adaptability, iTRAQ

Introduction

   Yak (Bos grunniens) is a unique and economically important animal on
   the Qinghai-Tibetan Plateau (QTP) ([43]1). Yaks are distributed in
   areas with low temperatures (average annual value ≤0°C), big day–night
   temperature difference (above 15°C), short growing season of herbages
   (110–135 d), high radiation intensity (annual radiation amount of
   140–195 KJ/cm^2), and low oxygen partial pressure (below 110 mm Hg)
   ([44]2). Due to the special ecological environment and strong natural
   selection, yaks have developed strong adaptability to natural
   environment conditions such as extreme cold, low oxygen, strong
   ultraviolet light, and nutrition stress in cold season ([45]3). In
   contrast, domestic cattle (Bos taurus) are raised in agricultural areas
   at low altitudes, with abundant feeding sources, where they can grow
   throughout the year ([46]4). In response to the harsh natural
   environment of the QTP, yaks have developed adaptive traits for
   long-term storage of nutrients and for managing hypoxia, including
   structural changes in internal organs and highly efficient oxygen
   transport ([47]5).

   The central nervous system of vertebrates is highly sensitive to
   hypoxia. As a long-term response to hypoxia, yaks possess special
   adaptive mechanisms. Even in the resting state, the brain requires high
   energy consumption and basic metabolism to maintain its normal
   functions ([48]6). Studies have shown that the brain is highly
   sensitive to environmental oxygen. Although the brain only accounts for
   2% of the total body weight, healthy brain tissue can obtain about 15%
   of the total cardiac output, and its oxygen consumption is as high as
   20% of the total oxygen consumption of the body ([49]7). How such a
   high oxygen utilization rate is achieved in the nervous system has
   remained a mystery. The oxygen delivered to brain tissue plays a
   crucial role in electrophysiological activities such as oxidative
   phosphorylation pathway, continuous ATP synthesis, and maintenance of
   sustainable energy supply ([50]8, [51]9). Previous studies have
   reported that the unique brain morphology of yaks can adapt to high
   altitude hypoxia ([52]10, [53]11). In addition, an analysis of yak
   mitochondrial genes revealed their associations with mitochondrial and
   oxidative phosphorylation, suggesting that the yak energy
   metabolism-related genes may change under natural pressure ([54]12,
   [55]13).

   Despite many studies on the plateau adaptability of yak, only a few
   have investigated the proteomic basis of this adaptation, including how
   modifications in aerobic respiration may contribute to efficient energy
   utilization. Being the site for the final oxidation of sugars, fats,
   and amino acids to release energy, mitochondria are the key organelles
   for oxidative metabolism in eukaryotic cells ([56]14). Mitochondria
   also function to regulate the cell cycle and cell growth and
   participate in cell differentiation, cell signaling, and apoptosis
   ([57]15). Characterization of the mitochondrial proteome of yaks may
   help to understand the molecular basis of their environmental
   adaptability.

   Yaks and cattle have a close evolutionary relationship; they belong to
   the same genus. Despite differences in their environmental habitats and
   physiology, there are similarities in their metabolic activities.
   Therefore, yak is considered as an appropriate model to explore the
   molecular mechanisms underlying plateau adaptations. We performed
   differential proteomic analysis using mitochondria extracted from the
   brain tissue of yaks and cattle. We identified differentially expressed
   proteins (DAPs) within the mitochondrial aerobic respiration pathway
   using isobaric tags for relative and absolute quantitation (iTRAQ)
   technology. We conducted a comparative proteomic analysis of yak and
   cattle brain mitochondrial proteins, identified DAPs and their related
   functions and pathways, and constructed protein–protein interaction
   networks. Based on our findings, we present a genetic mechanism for
   low-oxygen adaptation and provide new clues for understanding brain
   mitochondrial function.

Materials and Methods

Specimen Collection and Storage

   All yaks and cattle were handled in strict accordance with good animal
   practices that complied with the Animal Ethics Procedures and
   Guidelines of the People's Republic of China. The study was approved by
   the Animal Administration and Ethics Committee of Lanzhou Institute of
   Husbandry and Pharmaceutical Sciences of the Chinese Academy of
   Agricultural Sciences (Permit No. 2019-002).

   The yaks used in this study were obtained from the grazing area of the
   Ashidan Mountain region of Qinghai Province (altitude of 2,720 m),
   which is located in the Datong County, Qinghai Province. The Datong
   Breeding Farm in the Qinghai Province is located between a latitude of
   37° 11′-37° 32′ north and a longitude of 100° 52′-101° 26′ east in the
   southern foothills of Daji Mountain in the Qilian Mountain range.
   Cattle tissues used in this experiment were obtained from three healthy
   adult female Angus cattle. All cattle were from Angus Cattle Farm,
   Wuwei, Gansu Province at an altitude of 1,350 m. The yak and cattle
   investigated in this study were free grazing. The yak brain tissue was
   obtained from 3.5-year-old polled female yaks (n = 3). The cattle brain
   tissue was obtained from 3.5-year-old female cattle (n = 3). After
   animal sacrifice, the whole fresh brain from each animal was collected
   and washed with ice-cold phosphate-buffered saline (PBS). Brain tissues
   were cut in to small pieces, pulverized, mixed in liquid nitrogen, and
   stored at −80°C until use. Three individuals of yaks were named 113
   (yak-1), 114 (yak-2), and 116 (yak-3), while three individuals of
   cattle were named 118 (cattle-1), 119 (cattle-2), and 121 (cattle-3)
   ([58]Figure 1).

Figure 1.

   Figure 1
   [59]Open in a new tab

   The iTRAQ LC-MS/MS strategy for quantitative proteomic analysis of
   brain mitochondrial proteins in yak and cattle.

Mitochondrial Isolation and Protein Preparation

   Mitochondria were isolated using the ProteinExt Mammalian Mitochondria
   Isolation Kit for Tissue (Transgen Biotech, #DE501). The mitochondria
   obtained were assessed for their content and purity using 0.5% Janus
   Green B staining and light microscopy. Total protein was extracted from
   isolated mitochondria using the Protein Extraction Kit (Solarbio,
   #BC3710-50T). After removal of highly abundant proteins by three
   elutions, all protein samples were quantified using the bicinchoninic
   acid (BCA) method (Enhanced BCA Protein Assay Kit; Beyotime, #P0010S).

Proteolysis and Peptide Labeling

   Total protein (200 μg) per sample was adjusted to an equal volume (100
   μL) with 8 mol/L urea buffer. DL-dithiothreitol was added to each
   sample at a final concentration of 10 mmol/L and the samples were
   incubated at 37°C for 45 min. Following this, iodoacetamide was added
   to the sample at a final concentration of 25 mmol/L and the sample was
   incubated at room temperature for 55 min in the dark. Next, the sample
   was diluted with 100 mmol/L triethylammonium carbonate (TEAB) buffer
   until the urea concentration was <2 mol/L. Trypsin (Hualishi, China,
   #HLS TRY001C) was then added at a mass ratio of 1:50, and the sample
   mixture was digested overnight at 37°C. The next morning, trypsin was
   again added at a mass ratio of 1:100, and the sample mixture was
   further digested at 37°C for 4 h. Strata X C18 (Phenomenex) was used to
   desalt the protein sample after digestion, and the peptides were
   labeled according to the iTRAQ^® Reagent-8PLEX One Assay Kit
   (Sigma-Aldrich, #4381662). Briefly, peptides were dissolved in an
   appropriate volume of 100 mmol/L TEAB. Next, 41 μL of acetonitrile was
   reconstituted with 0.8 mg of iTRAQ reagent and combined with 100 μg of
   peptides for 1 h at room temperature, and hydroxylamine was added to
   terminate the labeling reaction. Next, the sample was vacuum dried. The
   yak samples were labeled with iTRAQ113, iTRAQ114, and iTRAQ116, while
   the cattle samples were labeled with iTRAQ118, iTRAQ119, and iTRAQ121.
   The labeled samples were then mixed and vacuum dried.

LC-MS/MS Analysis and Mass Spectrometry Data Search

   LC-MS/MS detection was performed on a hybrid quadrupole-time of flight
   LC-MS/MS mass spectrometer coupled with a nanospray source. Peptides
   were loaded onto a C18 trap column (Thermo Scientific, USA) and then
   eluted into a C18 analytical column (1.9 mm, 150 μm ×120mm, Thermo
   Scientific, USA). Three independent biological replicates were used for
   each sample, and peptides from these biological replicates were
   independently analyzed by the LC-MS/MS. Raw Thermo Scientific Q
   Exactive mass spectrometer was used to obtain the raw mass data in the
   RAW format. Thermo Proteome Discoverer 1.4 (Thermo Fisher, version
   1.4.0.288) was used to convert the RAW file into the MGF format.
   ProteinPilot (software version: 4.5) was used to analyze the mass
   spectroscopy data. After exporting the data to Excel, DEPs were
   screened according to the criteria of standard false discovery rate
   (FDR) <0.01. Any proteins with a differential score >1.3, a peptide
   number ≥2, a difference >1.5 (upregulated) or <0.67 (downregulated),
   and a P < 0.05 were selected as a DAP. The mass spectrometry proteomics
   data were deposited to the ProteomeXchange Consortium via PRIDE
   ([60]16) partner repository with the dataset identifier PXD024100.

Bioinformatics Analysis

   The bovine gene and protein databases were used as the alignment core.
   The DAPs identified in this study were cattle (Bos taurus) orthologous
   proteins. Protein information annotation, Gene Ontology (GO) analysis,
   and Reactome pathway analysis were performed using the ClusterProfiler
   package in R project [[61]http://www.r-project.org; ([62]17)], Uniprot
   protein database [[63]https://www.uniprot.org, UniProt release 2019_11;
   ([64]18)], and the Reactome database [[65]https://reactome.org/;
   ([66]19)]. Inter-protein interaction analysis was performed using
   STRING [[67]https://string-db.org, version 11; ([68]20)].

Western Blot Analysis

   To confirm the DAPs identified by iTRAQ, we randomly selected two DAPs
   (one upregulated and one downregulated). The primary antibodies used
   were rabbit anti-FKBP52 antibody (Beijing Biosynthesis Biotechnology,
   #bs-11270R) and anti-ATPAF2 antibody-middle region (Aviva Systems
   Biology, #ARP62162_P050) diluted to 1:1,200. The DAPs were probed with
   antibodies against glyceraldehyde-3-phosphate dehydrogenase (1: 3,000,
   Aviva Systems Biology, #OAAJ03302). The HRP-labeled anti-rabbit/mouse
   IgG (1:4,000, ProteinTech, #10285-1 AP) were used as secondary
   antibodies. Chemiluminescent detection was performed using Beyo ECL
   Star (Beyotime, #P0018,) and the ProteinSimple Multi-Function Imager
   was used for exposure, imaging, and densitometry. Western blotting was
   performed in triplicate and one-way analysis of variance (ANOVA) was
   performed using the R project.

Results

Identification of Differentially Abundant Proteins by iTRAQ

   To determine DAPs in mitochondria between yak and cattle, we utilized a
   proteomics approach based on iTRAQ quantification. In total, 4,517
   proteins were identified in the mitochondrial fraction samples, of
   which the global proteomic analysis detected a total of 370 proteins in
   yak and cattle brain tissue, each of which had at least two peptides
   identified with a minimum unused score of ≥1.3. This indicated a >95%
   confidence in correct sequence identification ([69]Supplementary Table
   1). Further analysis was performed on those proteins with a coefficient
   of variation <50%. Comparison of the mitochondrial proteomes identified
   57 proteins that met analysis criteria: average fold change of ≥1.5 or
   ≤ 0.67, and P < 0.05, of which 35 proteins were upregulated and 22 were
   downregulated in yak compared with cattle ([70]Supplementary Table 2).
   Four proteins were identified with a fold-change value lower than 0.5.
   These strongly downregulated proteins included short/branched chain
   specific acyl-CoA dehydrogenase (ACADSB), peptidylprolyl isomerase
   (FKBP4), phosphodiesterase (PDE2A), and pyrroline-5-carboxylate
   reductase 2 (PYCR2). The average fold-change of FKBP4 was the lowest
   (0.34). Eight proteins were identified with a fold-change value >2.0.
   These strongly upregulated proteins included aldehyde dehydrogenase
   (ALDH2), fumarate hydratase (FH), hydroxyacyl-CoA dehydrogenase (HADH),
   nucleolar protein 3 (NOL3), oxoglutarate dehydrogenase-like (OGDHL),
   cytochrome c oxidase subunit NDUFA4 (NDUFA4), sirtuin 2 (SIRT2), and
   isocitrate dehydrogenase (NADP) (IDH1). Among these, ALDH2 and NOL3
   were the most upregulated, with fold-change values of 3.05 and 2.77,
   respectively.

GO Analysis of Mitochondrial DAPs

   The GO definition was used to describe the functional concept of the
   gene and the relationship between DAPs (yak and cattle), including MF
   (molecular function), BP (biological process), and CC (cellular
   component) ([71]21). To identify GO functional groups enriched in DAPs,
   the GO database and genomic annotation information of cattle were used
   in the GO analyses. The 18 upregulated DAPs were significantly enriched
   (P < 0.01) for GO terms, including 6 MF terms, 8 BP terms, and 4 CC
   terms ([72]Figure 2A and [73]Supplementary Table 3). The 14
   downregulated DAPs were significantly enriched (P < 0.01) for GO terms,
   including 3 MF terms, 8 BP terms, and 3 CC terms ([74]Figure 2B). Among
   the upregulated DAPs, the enriched MF GO terms indicated that DAPs were
   mainly associated with NADH dehydrogenase (ubiquinone) activity, flavin
   adenine dinucleotide binding, and electron carrier activity. The top
   two GO terms were mitochondrion and mitochondrial matrix. Among the
   downregulated DAPs, the enriched GO terms included mitochondrion,
   oxidation–reduction process, and metabolic process.

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   GO classification of DAPs. (A) Upregulated proteins. (B) Downregulated
   proteins. The x-axis represents GO terms. The y-axis represents the
   number of enriched proteins within each primary category. MF, molecular
   function; BP, biological process; and CC, cellular component.

Pathway Enrichment Analysis of Mitochondrial DAPs

   To further explore the molecular mechanism underlying the plateau
   adaptability in yaks, we used the Reactome pathway database to assess
   the enrichment of metabolic pathways in the DAPs (yak vs. cattle). The
   enrichment results indicated that the 35 upregulated DAPs were involved
   in 12 pathways (FDR <0.05), including citric acid (TCA) cycle,
   respiratory electron transport, and pyruvate metabolism ([77]Figure 3A
   and [78]Table 1). Twenty-one downregulated DAPs included seven pathways
   (FDR <0.05), including metabolism, branched-chain amino acid
   catabolism, and mitochondrial beta-oxidation of unsaturated fatty acids
   ([79]Figure 3B and [80]Table 1). Therefore, the pathway analysis showed
   that the majority of the upregulated DAPs were related to the TCA cycle
   and respiratory electron transport, whereas the downregulated DAPs were
   related to mitochondrial fatty acid beta-oxidation and metabolism of
   amino acids and derivatives.

Figure 3.

   [81]Figure 3
   [82]Open in a new tab

   Reactome pathway analysis of DAPs. (A) Upregulated and (B)
   downregulated proteins in the mitochondria of yak brain tissue compared
   with that of cattle. The x-axis shows each of the enriched pathways.
   The y-axis represents the number of differentially abundant proteins
   (DAPs) in each primary category.

Table 1.

   Key pathways related to brain mitochondrial metabolism.
   Pathways FDR DAPs
   The citric acid (TCA) cycle and respiratory electron transport (up)
   2.97E10-11 NDUFA8, FH, NDUFA5, NDUFB5, NDUFA4, COX4I1, IDH1, ETFDH,
   ETFA, NDUFS8, NDUFAB1, ATP5D, D2HGDH
   Respiratory electron transport (up) 4.06E10-9 NDUFA8, NDUFS8, NDUFA5,
   NDUFB5, NDUFA4, COX4I1, NDUFAB1, ETFDH, ETFA
   Pyruvate metabolism and Citric TCA cycle (up) 0.031 FH, IDH1, D2HGDH
   Metabolism of amino acids and derivatives (down) 4.98E10-5 ALDH4A1,
   GSTZ1, AADAT, MCCC1, BCKDHB, PYCR2, PRODH, ACADSB
   Branched-chain amino acid catabolism (down) 0.002 MCCC1, BCKDHB, ACADSB
   mitochondrial fatty acid beta-oxidation of unsaturated fatty acids
   (down) 0.006 ECI1, ACADM
   [83]Open in a new tab

Functional Protein Association Networks of Mitochondrial DAPs

   The protein–protein interaction network of the DAPs identified in this
   study was analyzed using the Search Tool for the Retrieval of
   Interacting Proteins 11 (STRING 11) database. After removing
   unconnected proteins and self-loops, the PPI network identified protein
   nodes ([84]Figure 4). Proteins with higher connections than others in
   the network were designated key hubs, which may play significant roles
   in the network adjustment. Association analysis showed the top three
   key hub proteins to be ALDH4A1 (17 nodes), ALDH2 (15 nodes), and ECI1
   (15 nodes).

Figure 4.

   [85]Figure 4
   [86]Open in a new tab

   The protein–protein interaction network generated and visualized using
   STRING.

Verification and Analysis of Western Blots

   Western blot analysis was performed to verify the results from iTRAQ
   proteomics. Immunoblotting demonstrated that the expression of
   peptidylprolyl isomerase (FKBP4) in mitochondria from the brain tissue
   of yak was significantly lower than that found in cattle (P < 0.01;
   [87]Figure 5). The average ratio of FKBP4 expression in yak compared to
   that in cattle from western blotting was ~0.3026, which was slightly
   lower than the ratio 0.3433 from iTRAQ ([88]Supplementary Table 4). In
   contrast, the expression of ATP synthase mitochondrial F1 complex
   assembly factor 2 (ATPAF2) from the brain tissue of yak was
   significantly higher than that found in cattle (P < 0.01; [89]Figure
   5). The average ratio of ATPAF2 expression in yak compared to that in
   cattle from western blotting was ~1.2786 ([90]Supplementary Table 4),
   which was comparable to the ratio of 1.5034 from iTRAQ. Altogether,
   these data suggested that the immunoblotting results verified the
   results obtained from iTRAQ proteomics.

Figure 5.

   Figure 5
   [91]Open in a new tab

   Western blot of FKBP4 and ATPAF2.

Discussion

   Yak is a unique livestock species on the QTP, and its adaptation to
   harsh alpine conditions, including low oxygen partial pressure, high
   ultraviolet radiations, low grass quality, is much higher than that of
   its close relatives dwelling at low altitudes ([92]22). For grazing
   yaks, the efficient use of nutrients and energy is essential for
   survival through the long winter ([93]23). Previous studies have shown
   that the yak trachea, lungs, heart, and other organs are structurally
   adapted to plateau conditions ([94]24). Compared with their close
   relatives such as cattle, these changes include shortening of the
   trachea, increased thickness of the alveolar septum, thickening of the
   smooth muscle, and a higher blood cell density ([95]25). These
   structural adaptations bestow the yak with a high blood oxygen
   transport capacity and mitochondrial density, allowing efficient
   aerobic metabolism.

   Brain tissue, including the brain, cerebellum, and brainstem, is the
   highest energy-consuming tissue, regulating homeostatic body functions
   through neural activity. Considering the important functions of
   mitochondria, we sought to identify mitochondrial proteins that were
   significantly differentially expressed DAPs between yak and cattle, and
   conducted bioinformatics analysis on the identified proteins. Such DAPs
   may provide molecular insights into yak survival in the harsh plateau
   environment.

   Bioinformatics analysis of downregulated DAPs showed them to be
   implicated in several key pathways, such as metabolism of amino acids
   and their derivatives, as well as metabolism of branched amino acids
   ([96]Table 1). Valine, leucine, and isoleucine are branched-chain amino
   acids (BCAAs) that are important for protein synthesis and brain
   activity ([97]26). BCAAs, which are believed to enter the brain through
   the bloodstream and reduce the brain's production of serotonin, cause
   fatigue and reduced mental fatigue by decreasing serotonin levels
   ([98]27). Recent studies have shown that glutamate is available to both
   neurons, and astrocytes, and that when glutamate is present, astrocytes
   use glucose less, and ATP levels in astrocytes are higher when glucose
   is administered. The blood–brain barrier blocks the flow of glutamate
   into the brain; therefore, glutamate has little role in supporting
   brain energy production ([99]28). The upregulated DAP pathways
   suggested that the TCA cycle and respiratory electron transport are the
   most significant pathways (2.97E-11). A further molecular adaptation
   may be associated with the TCA cycle and respiratory electron
   transport. The TCA cycle is the converging metabolic pathway for the
   catabolism of three major nutrients (sugars, lipids, and amino acids)
   to release energy. As part of the oxidative breakdown of carbohydrates,
   FH hydrates fumarate to (S)-malate in the TCA cycle. Malic acid, the
   product of the TCA cycle, plays a key role in the downstream electron
   transfer and ATP synthesis ([100]29, [101]30). At low concentrations,
   ATP activates the heterotetramers and the heterodimer composed of IDH3A
   and IDH3G subunits; however, it inhibits their activities at high
   concentrations. In addition, ATP exhibits only an inhibitory effect on
   the heterodimer composed of IDH3A and IDH3B subunits ([102]31). In
   addition, malic enzyme (ME2) plays a significant role in the TCA
   pathway. It is involved in the de-acidification of malic acid, NAD
   binding, and metal ion binding, which could produce ATP to power
   cellular metabolic activity ([103]32, [104]33). Another potential
   source of molecular adaptation may be associated with the biogenesis of
   complex I. NADH dehydrogenase (NDUF) is a large protein comprising
   several protein sub-complexes ([105]34). Complex I, which is a
   secondary subunit of the mitochondrial membrane respiratory chain NADH
   dehydrogenase, does not participate in catalysis ([106]35). Complex I
   is involved in electron transfer from NADH to the respiratory chain.
   The direct electron acceptor of this enzyme is ubiquinone. Enhanced
   biogenesis of this complex may promote energy release through the
   electron transport chain. Overall, the upregulated DAPs suggest that
   yaks may have a highly efficient TCA cycle and energy utilization
   mechanism, allowing them to survive a long winter with nutrient
   shortage.

   The most enriched hub proteins were found to be ALDH4A1, ALDH2, and
   ECI1. ALDH is widely involved in the oxidation of aldehydes in the
   body. Excess aldehydes accumulate in the tissues and organs causing
   irreversible damage. ALDH4A1 and ALDH2 belong to the aldehyde
   dehydrogenase family. ALDH2 is located in the mitochondria and widely
   present in the human heart, brain, lungs, kidneys, liver, and other
   tissues. The ALDH2 gene in the ALDH2 acetaldehyde dehydrogenase family
   is the only one with high transcription in the brain ([107]36). The
   dehydrogenase activity of ALDH2 converts acetaldehydes to acetic acid,
   thereby reducing their cytotoxicity and protecting tissues and organs.
   Increasing the activity of ALDH2 protects again neurological damage
   ([108]37). In addition, hypoxia can increase lipid peroxidation causing
   increased lipid peroxidation products, including 4-HNE
   (4-hydroxynonenal) through the LO (lipoxygenase) pathway and ROS
   (reactive oxygen species) pathway. Studies have found that ALDH2
   regulates mitochondrial division through the 4-hydroxyenoic acid/HIF
   (hypoxia inducible factor)-1α/Drp1 (motility-related protein 1)
   signaling pathway ([109]37). ALDH4A1 can encode a
   pyrroline-5-carboxylic acid dehydrogenase involved in the degradation
   of proline, which catalyzes the conversion of pyrroline-5-carboxylic
   acid to glutamic acid ([110]38). ALDH4A1 deficiency leads to
   hyperprolinemia; patients present with epilepsy and intellectual
   disability ([111]39, [112]40). These proteins are mainly involved in
   bioenergy metabolism in hypoxic environments, suggesting that they may
   contribute to hypoxic adaptation mechanisms.

Conclusions

   In summary, this is the first study to provide proteomic data to
   support a molecular theory of plateau adaptability in yaks. We
   identified key DAPs; the majority of the upregulated DAPs were related
   to the TCA cycle and respiratory electron transport, whereas the
   downregulated DAPs were related to mitochondrial fatty acid
   beta-oxidation and metabolism of amino acids and derivatives. Hypoxia
   at high altitudes can regulate the activity of the mitochondrial
   respiratory chain. Changes in the mitochondrial structure and function
   are one of the important mechanisms in animals to overcome the hypoxic
   environment. Under a low oxygen environment, yak's endurance is
   enhanced as compared to that of cattle. We believe that these results
   will enhance our understanding of the underlying genetic and molecular
   mechanisms promoting plateau adaptability of yaks.

Data Availability Statement

   The datasets presented in this study can be found in online
   repositories. The names of the repository/repositories and accession
   number(s) can be found at: iProX; IPX0002625000.

Ethics Statement

   The animal study was reviewed and approved by all yaks and cattle
   involved in this study were in compliance with the guidelines for the
   care and use of laboratory animals issued by the State Council of the
   People's Republic of China. Additionally, the current investigation was
   approved by the Animal Management and Ethics Committee of the Lanzhou
   Institute of Animal Science and Veterinary Medicine, Chinese Academy of
   Agricultural Sciences (Permit No. SYXK-2016-0039).

Author Contributions

   PY and CL: conceptualization, project administration, resources, and
   supervision. XM and QZ: data curation, validation, and visualization.
   XM, QZ, and CL: formal analysis. XM and PY: investigation. DF, YL, HJ,
   PB, XW, and MC: methodology. XM: software and writing—original draft.
   XM, QZ, DF, HJ, PB, XW, MC, and XG: writing—review and editing. All
   authors contributed to the article and approved the, submitted version.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher's Note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

Footnotes

   Funding. This study was funded by the Agricultural Science and
   Technology Innovation Program (CAAS-ASTIP-2014-LIHPS-01), the Major
   Science and Technology Projects in Tibet Autonomous Region
   (XZ201901NA02), the National Beef Cattle Industry Technology & System
   (CARS-37), and the Herbivorous Livestock Industry and Technical System
   of Gansu Province (GARS08). Gansu Basic Research Innovation Group
   Program, Research on Innovation of Yak Molecular Breeding Technology
   (20JR5RA580), Special Fund for Basic Research Business Expenses of the
   Central-level Public Welfare Research Institutes (1610032021008), and
   the Science and Technology Aid Qinghai Cooperation Special Project
   (2020-QY-212) also supported this study.

Supplementary Material

   The Supplementary Material for this article can be found online at:
   [113]https://www.frontiersin.org/articles/10.3389/fvets.2021.663031/ful
   l#supplementary-material
   [114]Click here for additional data file.^ (1.5MB, XLSX)
   [115]Click here for additional data file.^ (25.9KB, XLSX)
   [116]Click here for additional data file.^ (15.6KB, XLSX)
   [117]Click here for additional data file.^ (13KB, XLSX)

References