Biology subjects

Pitarch, B.

Publications and source records attributed to Pitarch, B..

2 recordsLinked to original sources

bioRxiv2024

The structural permissiveness of triosephosphate isomerase (TpiA) of Escherichia coli

Triosephosphate isomerase (TpiA) is widely regarded as an example of an optimally evolved enzyme due to its essential role in biological systems, its structural conservation, and its near-perfect kinetic parameters. In this study, we investigated the structural robustness of the archetypal TpiA variant from Escherichia coli using an in vitro 5- amino acid linker scanning method. The resulting library was introduced into a tpiA mutant strain for functional complementation. From this library, 15 TpiA variants that were phenotypically indistinguishable from the wild-type enzyme were selected for further analysis. Although all variants retained enzymatic activities within the wild-type range, several insertions were found in highly structured protein domains where the linker was expected to cause significant structural perturbations. Despite these potentially disruptive additions, the enzymes maintained their activity even when expressed in a dnaK mutant, suggesting that chaperones did not compensate for structural abnormalities in vivo. Additionally, when these mutant TpiA variants were produced using the PURE in vitro transcription/translation system, they exhibited enzymatic activity comparable to, and in some cases exceeding, that of the non-mutated enzyme. AlphaFold2 revealed that insertions reconstructed the local architecture of the nearby amino acid sequences. The evolutionary implications of this remarkable structural resilience are discussed.

microbiology↗

bioRxiv2022

Bilallelic germline mutations in MAD1L1 induce a novel syndrome of aneuploidy with high tumor susceptibility

Aneuploidy is a frequent feature of human tumors. Germline mutations leading to aneuploidy are very rare in humans, and their tumor-promoting properties are mostly unknown at the molecular level. We report here novel germline biallelic mutations in MAD1L1, the gene encoding the Spindle Assembly Checkpoint (SAC) protein MAD1, in a 36-year-old female with a dozen of neoplasias, including five malignant tumors. Functional studies in peripheral blood cells demonstrated lack of full-length protein and deficient SAC response, resulting in [~]30-40% of aneuploid cells as detected by cytogenetic and single-cell (sc) DNA analysis. scRNA-seq analysis of patient blood cells identified mitochondrial stress accompanied by systemic inflammation with enhanced interferon and NFkB signaling. The inference of chromosomal aberrations from scRNA-seq analysis detected inflammatory signals both in aneuploid and euploid cells, suggesting a non-cell autonomous response to aneuploidy. In addition to random aneuploidies, MAD1L1 mutations resulted in specific clonal expansions of {gamma}{delta} T-cells with chromosome 18 gains and enhanced cytotoxic profile, as well as intermediate B-cells with chromosome 12 gains and transcriptomic signatures characteristic of chronic lymphocytic leukemia cells. These data point to MAD1L1 mutations as the cause of a new aneuploidy syndrome with systemic inflammation and unprecedented tumor susceptibility.

cancer biology↗