The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo.

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365-920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.

A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020.

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic.

After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.

The PPIP5K Family Member Asp1 Controls Inorganic Polyphosphate Metabolism in S. pombe.

Inorganic polyphosphate (polyP) which is ubiquitously present in both prokaryotic and eukaryotic cells, consists of up to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this polymer is manifold and diverse and in fungi ranges from cell cycle control, phosphate homeostasis and virulence to post-translational protein modification. Control of polyP metabolism has been studied extensively in the budding yeast Saccharomyces cerevisiae. In this yeast, a specific class of inositol pyrophosphates (IPPs), named IP7, made by the IP6K family member Kcs1 regulate polyP synthesis by associating with the SPX domains of the vacuolar transporter chaperone (VTC) complex. To assess if this type of regulation was evolutionarily conserved, we determined the elements regulating polyP generation in the distantly related fission yeast Schizosaccharomyces pombe. Here, the VTC machinery is also essential for polyP generation. However, and in contrast to S. cerevisiae, a different IPP class generated by the bifunctional PPIP5K family member Asp1 control polyP metabolism. The analysis of Asp1 variant S. pombe strains revealed that cellular polyP levels directly correlate with Asp1-made IP8 levels, demonstrating a dose-dependent regulation. Thus, while the mechanism of polyP synthesis in yeasts is conserved, the IPP player regulating polyP metabolism is diverse.

Asp1 Bifunctional Activity Modulates Spindle Function via Controlling Cellular Inositol Pyrophosphate Levels in Schizosaccharomyces pombe.

The generation of two daughter cells with the same genetic information requires error-free chromosome segregation during mitosis. Chromosome transmission fidelity is dependent on spindle structure/function, which requires Asp1 in the fission yeast Schizosaccharomyces pombe Asp1 belongs to the diphosphoinositol pentakisphosphate kinase (PPIP5K)/Vip1 family which generates high-energy inositol pyrophosphate (IPP) molecules. Here, we show that Asp1 is a bifunctional enzyme in vivo: Asp1 kinase generates specific IPPs which are the substrates of the Asp1 pyrophosphatase. Intracellular levels of these IPPs directly correlate with microtubule stability: pyrophosphatase loss-of-function mutants raised Asp1-made IPP levels 2-fold, thus increasing microtubule stability, while overexpression of the pyrophosphatase decreased microtubule stability. Absence of Asp1-generated IPPs resulted in an aberrant, increased spindle association of the S. pombe kinesin-5 family member Cut7, which led to spindle collapse. Thus, chromosome transmission is controlled via intracellular IPP levels. Intriguingly, identification of the mitochondrion-associated Met10 protein as the first pyrophosphatase inhibitor revealed that IPPs also regulate mitochondrial distribution.