Biology subjects

Calabrese, G.

Publications and source records attributed to Calabrese, G..

6 recordsLinked to original sources

bioRxiv2026

Chaperoning through time - defining DNAJA2 co-chaperone client selectivity via pulse-SILAC and BioID.

Molecular chaperones are a major driving force ensuring efficient protein folding and regulating proteostasis in the cell. However, it remains unclear how their clients are selected in most cases, especially after the release of nascent protein chains from ribosomes. Here, we present a novel approach that combines pulse metabolic labelling with SILAC and BioID mass spectrometry to temporally resolve protein-protein interactions of a co-chaperone. Using the Hsp70 co-chaperone DNAJA2 as a benchmark, we reveal that two distinct pools of proteins are enriched. In particular, DNAJA2 displays a preferential association with highly structured recently synthesized proteins enriched in {beta}-strands. In contrast, pre-existing proteins captured in our assay exhibit higher intrinsic disorder. In both cases, these proteins tend to be longer and contain a lower net charge compared to the proteome. Notably, these preferential associations are retained upon heat-shock, while interactions with the "older" pool of proteins become more prevalent under these conditions. Through this methodology, we gain novel insights on how co-chaperone-client interactions may occur over the lifespan of a protein to preserve proteostasis.

biochemistry↗

bioRxiv2024

Tsa1 is the dominant peroxide scavenger and a source of H2O2-dependent GSSG production in yeast

Hydrogen peroxide (H2O2) is an important biological molecule, functioning both as a second messenger in cell signaling and, especially at higher concentrations, as a cause of cell damage. Cells harbor multiple enzymes that have peroxide reducing activity in vitro. However, the contribution of each of these enzymes towards peroxide scavenging in vivo is less clear. Therefore, to directly investigate in vivo peroxide scavenging, we used the genetically encoded peroxide sensors, roGFP2-Tsa2{Delta}CR and HyPer7, to systematically screen the peroxide scavenging capacity of yeast thiol and heme peroxidase mutants. We show that the 2-Cys peroxiredoxin Tsa1 alone is responsible for almost all exogenous H2O2 and tert-butyl hydroperoxide scavenging. The two catalases and cytochrome c peroxidase only produce observable scavenging defects at higher H2O2 concentrations when these three heme peroxidases are deleted in combination. We also analyzed the reduction of Tsa1 in vitro, revealing that the enzyme is efficiently reduced by thioredoxin 1 with a rate constant of 2.8x106 M-1s-1. When thioredoxins are oxidized, Tsa1 can become an important source of H2 O2 -dependent cytosolic glutathione disulfide production in yeast. Our findings clarify the importance of the various thiol and heme peroxidases for peroxide removal and suggest that most thiol peroxidases have alternative or specialized functions in specific subcellular compartments.

biochemistry↗

bioRxiv2024

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

Mutations can result in the loss of a proteins native function due to protein misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors of one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant protein but not of the wild-type protein. Our work indicates that missense mutations in cytosolic proteins can promote interactions with molecular chaperones that normally do not occur. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits three distinct behaviours: acting to stabilize solely the mutant, both the wild-type and mutant proteins, or being dispensable. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein. Summary StatementWe identified a panel of cytosolic mutant proteins degraded by the proteasome. DNAJA2 is often required to prevent mutant protein turnover, even if it is sometimes dispensable for the wild-type protein.

cell biology↗

bioRxiv2022

Bacterial aerobic methane cycling by the marine sponge-associated microbiome

BackgroundMethanotrophy by the sponge-hosted microbiome has been mainly reported in the ecological context of deep-sea hydrocarbon seep niches where methane is either produced geothermically or via anaerobic methanogenic archaea inhabiting the sulfate-depleted sediments. However, methane oxidizing bacteria from the candidate phylum Binatota have recently been described and shown to be present in oxic shallow-water marine sponges, where sources of methane remain undescribed. ResultsHere, using an integrative -omics approach, we provide evidence for sponge-hosted bacterial methanogenesis occurring in fully oxygenated shallow water habitats. Specifically, we suggest methanogenesis occurs via at least two independent pathways involving methylamine and methylphosphonate transformations that, concomitantly to aerobic methane production, generate bioavailable nitrogen and phosphate, respectively. Methylphosphonate may be sourced from seawater continuously filtered by the sponge host. Methylamines may also be externally sourced or, alternatively, generated by a multi-step metabolic process where carnitine, derived from sponge cell debris, is transformed to methylamine by different sponge-hosted microbial lineages. Finally, methanotrophs specialized in pigment production, affiliated to the phylum Binatota, may provide a photoprotective function, closing a previously undescribed C1-metabolic loop that involves both the sponge host and specific members of the associated microbial community. ConclusionGiven the global distribution of this ancient animal lineage and their remarkable water filtration activity, sponge hosted methane cycling may affect methane supersaturation in oxic coastal environments. Depending on the net balance between methanogenesis and methanotrophy, sponges may serve as marine sources or sinks of this potent greenhouse gas.

microbiology↗

bioRxiv2021

Transcriptome-wide Association Study and eQTL colocalization identify potentially causal genes responsible for bone mineral density GWAS associations

Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni <= 0.05) using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.

genomics↗

bioRxiv2019

Genetic Analysis of Osteoblast Activity Identifies Zbtb40 as a Regulator of Osteoblast Activity and Bone Mass

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

genetics↗