Biology subjects

Giacomelli, A. O.

Publications and source records attributed to Giacomelli, A. O..

4 recordsLinked to original sources

bioRxiv2024

Dominant-negative TP53 mutations potentiated by the HSF1-regulatedproteostasis network

Protein mutational landscapes are shaped by how amino acid substitutions affect stability and folding or aggregation kinetics. These properties are modulated by cellular proteostasis networks. Heat shock factor 1 (HSF1) is the master regulator of cytosolic and nuclear proteostasis. Chronic HSF1 ac-tivity upregulation is a hallmark of cancer cells, potentially because upregulated proteostasis factors fa-cilitate the acquisition and maintenance of oncogenic mutations. Here, we assess how HSF1 activation influences mutational trajectories by which p53 can escape cytotoxic pressure from nutlin-3, an inhibitor of the p53 regulator MDM2. HSF1 activation broadly increases the fitness of dominant-negative p53 substitutions, particularly non-conservative, biophysically unfavorable amino acid changes within buried regions of the p53 DNA-binding domain. These findings demonstrate that HSF1 activation reshapes the oncogenic mutational landscape by preferentially supporting the emergence and persistence of bio-physically disruptive, cancer-associated p53 substitutions, linking proteostasis network activity directly to oncogenic evolution.

molecular biology↗

bioRxiv2024

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRASG12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRASG12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

cancer biology↗

bioRxiv2023

RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4

Microsatellite instability high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion, cell proliferation, and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

cancer biology↗

bioRxiv2021

Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

Pooled variant expression libraries can test the phenotypes of thousands of variants of a gene in a single multiplexed experiment. In a library encoding all single-amino-acid substitutions of a protein, each variant differs from its reference only at a single codon-position located anywhere along the coding sequence. Consequently, accurately identifying these variants by sequencing is a major technical challenge. A popular but expensive brute-force approach is to divide the pool of variants into multiple smaller sub-libraries that each contains variants of a small region and that must each be constructed and screened individually, but that can then be PCR-amplified and fully sequenced with a single read to allow direct readout of variant abundance. Here we present an approach to screen very large variant libraries with mutations spanning a wide region in a single pool, including library design criteria and mutant-detection algorithms that permit reliable calling and counting of variants from large-scale sequencing data.

systems biology↗