No abstract

The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In T. gondii and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.

23The mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for 24 maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, 25 many subunit components, necessary for proper assembly and functioning of this enzyme, 26 appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-27 type ATP synthase from mass spectrometry analysis of partially purified monomeric (~600 28 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, 29 key F O subunits, a, b and d, can be identified from conserved structural features. Orthologs 30 for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. 31Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit 32 composition of this enzyme, within the alveolate clade. Discovery of these highly diversified 33 novel components of the apicomplexan F-type ATP synthase complex could facilitate the 34 development of novel anti-parasitic agents. Structural and functional characterization of this 35 unusual enzyme complex will advance our fundamental understanding of energy metabolism 36 in apicomplexan species. 37 38 39 40 41 42 43 44 45 46 47the presence of glutamine as an alternative nutrient source [35,36]. In the absence of glucose 98 oxidation via glycolysis, oxidative phosphorylation is the only source for bulk cellular ATP. 99Evidence for an active oxidative phosphorylation in T. gondii exists [37][38][39], and treatment 100 with atovaquone, a potent inhibitor of mitochondrial electron transport chain (mtETC), 101 results in inhibition of ATP synthesis and parasite growth (EC 50 < 10 nM) [38]. 102 Very little is known about the structure and function of the F-type ATP synthase from 103 T. gondii and other apicomplexan parasites. Comparative genomics has revealed that, while 104 the five canonical F 1 sector subunitsα, β, γ, δ, and ε -are readily identified in all 105 apicomplexan parasites based on amino acid sequence conservation, only two F O sector 106 subunits -c and OSCP -can be identified by sequence [25-27]. Characterization of F-type 107 ATP synthase from solubilized mitochondria lysates of the human malaria parasite 108 Plasmodium falciparum revealed that the enzyme assembles into monomer and dimer forms 109 [26]. This suggests that a full complement of subunits, typical for the eukaryotic enzyme, is 110 present in P. falciparum and likely in other apicomplexan parasites as well. 111 Here, we have investigated the subunit composition of the enzyme from T. gondii. We 112 were able to identify and partially purify the F-type ATP synthase enzyme complex from 113 solubilized mitochondrial preparation of T. gondii using blue native PAGE separation, by 114 immuno-precipitation and by chromatographic enrichment. LC-MS analysis of the enzyme 115 preparations revealed the identity of the proteins associated with the T. gondii F-type ATP 116 synthase. We have identified 20 novel proteins (of unknown func...

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. The availability of patient hospital records is crucial for linking the genomic sequence information to virus function during the course of infections. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. From the assembled sequences, we estimate the SARS-CoV-2 effective population size and infection rate and outline the epidemiological dynamics of import and transmission events during this period in Saudi Arabia. We show that two consecutive mutations (R203K/G204R) in the SARS-CoV-2 nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein by mass-spectrometry analysis. Furthermore, analysis of the host cell transcriptome suggests that the mutant N protein results in dysregulated interferon response genes. We provide crucial information in linking the R203K/G204R mutations in the N protein as a major modulator of host-virus interactions and increased viral load and underline the potential of the nucleocapsid protein as a drug target during infection.