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Post by Admin on Jun 19, 2021 23:13:04 GMT
Skoltech scientists and their colleagues from Germany and the United States have analyzed the metabolomes of humans, chimpanzees, and macaques in muscle, kidney, and three different brain regions. The team discovered that the modern human genome undergoes mutation which makes the adenylosuccinate lyase enzyme less stable, leading to a decrease in purine synthesis. This mutation did not occur in Neanderthals, so the scientists believe that it affected metabolism in brain tissues and thereby strongly contributed to modern humans evolving into a separate species. The research was published in the journal eLife. The predecessors of modern humans split from their closest evolutionary relatives, Neanderthals, and Denisovans, about 600,000 years ago, while the evolutionary divergence between our ancestors and those of modern chimpanzees dates as far back as 65 million years ago. Evolutionary biologists are after the particular genetic features that distinguish modern humans from their ancestors and may give a clue as to why humans are what they are. Researchers from the Skoltech Center for Neurobiology and Brain Restoration (CNBR) led by Professor Philipp Khaitovich and their colleagues from the Max Planck Institutes in Leipzig, Dresden and Cologne and the University of Denver studied metabolic differences in the brain, kidney, and muscle of humans, chimpanzees, and macaques. The research supervisor was a renowned evolutionary biologist, Professor Svante Pääbo, who earlier on had discovered the Denisovan and led the Neanderthal Genome Project. The team looked at an interesting human mutation that leads to amino acid substitution in adenylosuccinate lyase, an enzyme involved in the synthesis of purine inside DNA. This substitution reduces the enzyme’s activity and stability, which results in a lower concentration of purines in the human brain. The team showed that the new mutation is typical for humans only and does not appear in other primates or Neanderthals. The researchers proved that this mutation is indeed the reason for the metabolic peculiarities in humans by introducing it into the mouse genome. The mice subjected to mutation produced fewer purines, whereas an ancestral gene, when introduced into human cells, led to apparent metabolic changes. “Although a powerful tool for scientists, the decoded human genome, unfortunately, cannot account for all the phenotypic differences between humans. The study of the metabolic composition of tissues can give clues about why functional changes occur in humans. I am delighted that we have succeeded in predicting the metabolic characteristics of modern humans and validated our hypotheses on mouse and cell models, even though we did not have ‘live Neanderthals’ to work on,” says lead author and Skoltech PhD student Vita Stepanova. Reference: “Reduced purine biosynthesis in humans after their divergence from Neandertals” by Vita Stepanova, Kaja Ewa Moczulska, Guido N Vacano, Ilia Kurochkin, Xiangchun Ju, Stephan Riesenberg, Dominik Macak, Tomislav Maricic, Linda Dombrowski, Maria Schörnig, Konstantinos Anastassiadis, Oliver Baker, Ronald Naumann, Ekaterina Khrameeva, Anna Vanushkina, Elena Stekolshchikova, Alina Egorova, Anna Tkachev, Randall Mazzarino, Nathan Duval, Dmitri Zubkov, Patrick Giavalisco, Terry G Wilkinson, David Patterson, Philipp Khaitovich and Svante Pääbo, 4 May 2021, eLife. DOI: 10.7554/eLife.58741
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Post by Admin on Jun 20, 2021 20:26:54 GMT
Reduced purine biosynthesis in humans after their divergence from Neandertals Vita Stepanova Is a corresponding author et al. Research Article May 4, 2021
Abstract We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.
Introduction Modern humans differ dramatically from their closest evolutionary relatives in a number of ways. Most strikingly, they have developed rapidly changing and complex cultures that have allowed them to become more numerous and widespread than any other primates and closely related hominins such as Neandertals. This unique historical development has at least to some extent biological roots. However, although large numbers of traits have been identified as being unique to humans or been suggested to be so (e.g. Tomasello, 2019, Varki and Altheide, 2005), it has proven difficult to identify the genetic and biological underpinnings of such traits. One reason may be that many of them are genetically complex (Phillips, 2008) and represent differences in degree rather than absolute differences between humans and other groups. In particular, it is unclear if any single nucleotide substitution or other genomic change that occurs in all or almost all humans today, but is not seen among the closest evolutionary relatives of present-day humans, the Neandertals, and Denisovans (Pääbo, 2014), has biological consequences. A duplicative transposition in modern humans of the gene BOLA2 which is involved in iron metabolism has been described (Nuttle et al., 2016) in a region associated with autism but the functional consequences are unknown. A recent study introduced a modern human-specific amino acid substitution in the splice factor NOVA1 and suggested that it has drastic effects on the differentiation of brain organoids (Trujillo et al., 2021), an observation that warrants further study.
To identify traits that may have comparatively simple genetic architectures yet influence the biology of modern humans, we focus here on metabolic differences. By analyzing the metabolomes of muscle, kidney and three different regions of the brain from humans, chimpanzees and macaques, we find that many aspects of amino acid metabolism differ between humans and the other two primates in all tissues analyzed. Among metabolic pathways, purine biosynthesis is less active in several human tissues including the brain. In purine biosynthesis, we find that metabolites downstream of the enzyme adenylosuccinate lyase [ADSL (EC 4.3.2.2)] occur at lower concentrations in humans than in the other primates. ADSL carries an amino acid substitution that is unique to modern humans relative to apes and Neandertals and Denisovans (Castellano et al., 2014) and has been shown to affect the stability of the enzyme (Van Laer et al., 2018). By introducing the modern human-like substitution in the genome of mice, and the ancestral, Neandertal-like substitution in the genomes of human cells, we show that this substitution contributes to much or all of this metabolic change in present-day humans.
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Post by Admin on Jun 21, 2021 5:07:38 GMT
Results Metabolite changes unique to humans To identify metabolites that have changed their concentration in humans relative to monkeys and apes, we measured compound concentrations in prefrontal cortex, primary visual cortex, cerebellum, skeletal muscle and kidney in four humans, four chimpanzees, and four macaques using mass spectrometry coupled with capillary electrophoresis (CE-MS), a technique suitable for detection of small hydrophilic compounds (Figure 1a,b). The number of metabolites annotated in the five tissues varied between 166 and 197, 160 and 209, and 145 and 192 in the three species, respectively (Supplementary file 1). Figure 1 Primate metabolome analysis. (a) Sketch depicting species and tissues used for metabolite measurements. Red arrows indicate sampled brain regions: the dorsolateral prefrontal cortex, primary visual cortex, and cerebellar cortex. (b) Multidimensional scaling (MDS) analysis of all samples (left) and brain samples (right) based on the intensities of all detected metabolic peaks. (c) An analysis of KEGG-based metabolic pathway showing for each pathway (circle) the number of detected metabolites (X axis) and the cumulative p-value of the difference (Y axis) between human and non-human primate (chimpanzee and macaque) intensity levels within the pathway in the five tissues. Left: an analysis based on metabolites with lower intensity in humans compared to non-human primates. The oxidative phosphorylation pathway is the second highest. Right: an analysis based on metabolites with higher intensity in humans. The size of the symbols is proportional to the -logarithm-transformed p-value. The dotted line indicates nominal p=10−10. In each tissue, we identified metabolites that did not differ significantly with respect to their mass spectrometric peak intensities between the macaques and the chimpanzees, but differed significantly, and in the same direction, between humans and macaques as well as between humans and chimpanzees. Whereas we find no such metabolites in skeletal muscle and kidney, we find metabolites peak intensities and thus concentrations differ in humans relative to the other two primates in two of the three parts of the brain analyzed. In cerebellum, 22 metabolites have higher concentrations in humans whereas no metabolites have lower concentrations (Supplementary file 2). Eighteen of the 22 metabolites are amino acids. In prefrontal cortex, we detected five metabolites with lower concentrations in humans and none with higher concentrations (Supplementary file 3). Three of the five metabolites are purines (inosine monophosphate [IMP], guanosine monophosphate [GMP], adenosine monophosphate [AMP]) and the other two are NAD+ and UDP-N-acetylglucosamine. Metabolic pathways To identify metabolic pathways that may be more or less active in humans than in other primates, we linked metabolites with higher or lower concentrations in humans compared to both chimpanzees and macaques (even if not significantly so) to genes and metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Supplementary files 4 and 5 give pathways that contain more genes than expected (p<0.01) that are associated with metabolites present in either higher or lower concentrations in humans in each of the five tissues. Between 8 and 12 pathways are associated with higher metabolite concentrations in the five human tissues (Supplementary file 4). All eight pathways identified in the prefrontal cortex involve amino acid metabolism, eight out of nine pathways in the visual cortex and nine out of 10 in the cerebellum also involve amino acids. In muscle and kidney, 7 and 5 of the 11 and 12 pathways identified, respectively, involve amino acid metabolism. Thus, several aspects of amino acid metabolism seem to be increased in humans relative to other primates. Between 4 and 11 pathways are associated with lower metabolite concentrations in the five tissues (Supplementary file 5). Amino acid and peptide metabolism make up one to five of these pathways in the different tissues, suggesting that amino acid metabolism has changed in several ways in humans relative to the other primates. Hence, metabolites in these pathways are present in increased as well as decreased concentrations, often in the same pathways. For example, significant numbers of metabolites in arginine and proline metabolism are present in higher concentrations while other metabolites in the same pathways are present in lower concentrations, sometimes in the same tissues, suggesting that the flux through these pathways has changed. Metabolites in two pathways are consistently present at lower rather than higher concentrations in humans. One of these pathways is oxidative phosphorylation, which shows decreased metabolite concentrations in the three brain regions. Because oxidative phosphorylation is not affected in muscle and kidneys, it seems that the human brain differs from ape brains in that oxidative phosphorylation in mitochondria is less active. This may deserve future investigation. The second pathway that stands out is purine biosynthesis where a number of metabolites are present at lower levels in humans (Figure 1c). Based on the KEGG definition of the pathway, purine biosynthesis is decreased in humans relative to apes in the brain as well as in other organs. If we focus on a more restrictive definition of purine biosynthesis (Marie et al., 2004), a significant decrease is seen in the three brain regions and not in muscle and kidney (Figure 2). Further, three of the five metabolites, IMP, GMP and AMP, which individually have significantly lower concentrations in humans, are products of de novo purine biosynthesis. Taken together, metabolites involved in purines biosynthesis have significantly lower concentrations in humans than in non-human primates in all three brain regions than do metabolites in the other metabolic pathways, (Wilcoxon test, p=0.049 (PFC), p=0.002 (CB), p=0.001 (VC); Figure 2c). Similarly, we find the significantly lower concentrations of purine biosynthesis metabolites in humans compared to chimpanzees and macaques relative to other metabolic pathways when analyzing previously published metabolite data from the PFC of three species (Kurochkin et al., 2019) (Wilcoxon test, p=0.019; Figure 2d). Thus, purine biosynthesis stands out as down-regulated in humans. This is seen in all tissues but is particularly pronounced in the brain.
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Post by Admin on Jun 21, 2021 21:38:19 GMT
Figure 2 Purine metabolite levels in humans relative to chimpanzees and macaques. (a) Pathway sketch illustrating the role of ADSL in purine biosynthesis. (b) Differences in metabolite levels between humans (n = 4) and chimpanzees (C) (n = 4), macaques (M) (n = 4) and the two non-human primates combined (P) in prefrontal cortex (PFC), cerebellum (CB), primary visual cortex (VC) analyzed by CE-MS. LC-MS measurements in PFC are from Kurochkin et al., 2019 and involved 40 humans, chimpanzees and macaques, respectively. Red and blue colors indicate higher and lower levels in humans, respectively. c and d. The distributions of metabolite differences between humans and non-human primates for purines biosynthesis (PB) (listed in panel b) and other metabolites (non-PB) detected by CE-MS (c) and LC-MS (d). The Y axis shows fold-change difference between humans and non-human primates. The metabolite concentration values are normalized and log-transformed. Wilcoxon tests for the differences between PB and non-PB distributions: p=0.049 (PFC), p=0.002 (CB), p=0.001 (VC), and p=0.019 (PFC LC-MC). Adenylosuccinate lyase (ADSL) in modern humans The enzyme adenylosuccinate lyase (ADSL) catalyzes two reactions in de novo purine biosynthesis (Figure 2a; Ciardo et al., 2001; Jaeken and Van den Berghe, 1984). First, it cleaves succinylaminoimidazole carboxamide ribotide (SAICAR) into aminoimidazole carboxamide ribotide (AICAR) and fumarate. Second, it cleaves adenylosuccinate (S-AMP) into adenosine monophosphate (AMP) and fumarate (Figure 2a). The compounds AMP, IMP, and GMP that are reduced in the three human brain regions analyzed are situated downstream of ADSL, consistent with the hypothesis that a change in ADSL may have caused the reduction in purine biosynthesis. ADSL (Ariyananda et al., 2009) is a homotetrameric complex where three monomers contribute to each of the four active sites (Brosius and Colman, 2002). The gene encoding ADSL in humans is located on chromosome 22q13.1–13.2 and is one of only approximately one hundred protein-coding genes that carry substitutions inferred to change amino acids that are fixed or almost fixed in present-day humans but occur in their ancestral, ape-like state in the genomes of the closest extinct relatives of modern humans, Neandertals and Denisovans (Pääbo, 2014). The substitution in ADSL in modern humans is an alanine to valine substitution at position 429 (A429V). Neandertals, Denisovans, all other primates and most mammals carry an alanine residue at this position (Figure 3). The substitution is located in a protein domain that forms part of the substrate channel over the active site of the enzyme. Amino acid substitutions close to this position cause lowered enzymatic activity and/or lower enzyme stability and result in symptoms that include psychomotor retardation, autism, and epilepsy (Jurecka et al., 2015; Spiegel et al., 2006; Zikanova et al., 2010) as well as alterations in brain structure as seen by magnetic resonance imaging (Jurecka et al., 2012). This raises the possibility that the amino acid substitution at position 429 may have changed the activity of ADSL in modern humans after their separation from the lineage leading to Neandertals and Denisovans. Figure 3 ADSL protein sequence. Partial amino acid sequences of ADSL. Accession numbers and amino acid sequences are from NCBI. X-ray structural determination of ADSL (DOI: 10.2210/pdb4FFX/pdb) is from Ray et al., 2012. Arrow … see more A mouse humanized for Adsl To investigate how the A429V substitution may affect purine biosynthesis, we introduced this mutation into the Adsl gene of a C57BL/6N mouse by injection of the relevant oligonucleotide and CRISPR-Cas9 into the male pronucleus of fertilized oocytes. Adjacent to the amino acid of interest, at position 428, mice carry an arginine residue whereas primates carry a glutamine residue (Figure 3). To avoid possible effects of the rodent-specific arginine residue at position 428 on the function of the amino acid at position 429, we introduced a nucleotide substitution resulting in an R428Q substitution in addition to the V429A substitution in the Adsl gene. The two mutations segregate in Mendelian ratios in the mice, and animals heterozygous and homozygous for the two substitutions show no overt phenotypic difference to their wild-type littermates.
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Post by Admin on Jun 22, 2021 0:41:07 GMT
ADSL activity in the humanized mice We analyzed the enzymatic activities of ADSL in tissue extracts prepared from mice homozygous for the human-like mutations and their wild-type littermates in cerebellum, cerebral cortex, heart, kidney, liver, lung, muscle, spleen, and testis in 12-week-old mice and (except testis) in 1-week-old mice by measuring the conversion of adenylosuccinate to AMP. In the wild-type adult mice (n = 10), mean ADSL activity varied from 1.95 nmole/min/μg in liver to 24.4 nmole/min/μg in muscle (Figure 4b). In the pups (n = 10), mean enzymatic activity was 5.4% to 57% higher in most tissues and largely paralleled those of the adults (Figure 4a). Two exceptions were cerebellum and muscle, where ADSL activity was 41% and 11% lower in the pups than in the adults, respectively. Figure 4 ADSL activity in tissues from humanized and wild type mice. (a) One-week-old mice. (b) Twelve-week-old mice. Conversion of adenylosuccinate to AMP was monitored by measuring λ 282 nm absorbance over 20 min (see Materials and methods). Boxes represent upper and lower quartiles, lines extending from the boxes represent the ranges of the data, excluding outliers which are indicated by dots. In the humanized mice, ADSL activity was reduced by 8.7% to 56% in all organs in both adults (n = 11) and pups (n = 12) (Figure 4; p<0.05, for all comparisons in both age groups). The relative reduction of the activity in the humanized relative to the wild-type mice is higher in cerebral cortex (43% in pups and 56% in adults) than in the other tissues (8.7%–40%). Thus, young as well as adult mice that carry an ADSL enzyme humanized at positions 428 and 429 have lower ADSL activity in most or all tissues.
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