Antonis Kirmizis

The development of a living End-to-End (E2E) testbed for investigating and validating Molecular Communication (MC) at the microscale, remains a critical challenge. In this paper, we address this challenge, by reporting ongoing work towards the development and mathematical characterization of a "transmitter" cell, based on the yeast model organism, which can complement our previously developed "receiver" cell, for delivering a complete living E2E system. The significance of such a testbed stems from the genetic amenability of yeast which can facilitate future research activities as well as the high relevance to emerging biomedical applications. Yeast cells have been recently leveraged for the development of relevant biosensors for biomarker detection and information dissemination, being explored for their potential to act as MC receivers when integrated on ingestible smart capsules, while their integration on Organs-On-Chip underpins their role as transmitters relaying locally sensed data to information collection devices.

Glioblastoma (GBM) is the most aggressive and lethal form of primary brain cancer in adults, characterized by rapid cell proliferation, resistance to therapy, and poor patient outcomes. While epigenetic dysregulation is increasingly recognized in glioma pathogenesis, the role of Histone Acetyltransferase 1 (HAT1) in GBM remains largely undefined. Here, we demonstrate that HAT1 expression is progressively reduced in high-grade gliomas of human tissue microarrays as well as in glioma cell lines with increased malignancy. We show that HAT1 depletion in glioma cells significantly enhances cell proliferation and membrane fluidity specifically in aggressive GBM subtypes (A172 and U87). Integrated transcriptomics and lipidomics analyses revealed upregulation of lipid metabolism pathways, including enhanced glycerophospholipid biosynthesis, alongside a marked induction of aldehyde dehydrogenase 2 (ALDH2) expression in HAT1-deficient cells. In contrast, HAT1 re-expression suppressed proliferation, restored membrane properties, and downregulated ALDH2. Subcutaneous xenograft models confirmed that HAT1 loss accelerates tumor growth, while HAT1 overexpression restrains it. Notably, knockdown of ALDH2 in HAT1-depleted GBM cells reversed the hyperproliferative and high-membrane-fluidity phenotype, identifying ALDH2 as a key downstream effector. Collectively, our data uncover a novel HAT1–ALDH2–lipid remodeling axis that regulates GBM cell proliferation and membrane plasticity, positioning HAT1 as a suppressor of GBM aggressiveness and highlighting this pathway as a potential axis for prognostic and therapeutic exploitation in GBM.

N-terminal acetylation is an abundant and predominantly co-translational modification in eukaryotes that profoundly affects folding, compartmentalization fidelity and turnover of target proteins. Unlike other N-acetyltransferases, human NatD is composed solely of the catalytic subunit NAA40 and exclusively modifies histone proteins H2A and H4. However, the molecular details of co-translational NAA40 activity have remained elusive. Here, we show biochemically and by cryo-EM how NAA40 activity is coordinated at the ribosomal peptide tunnel exit involving the NAC complex. We demonstrate that the NAA40-NAC interaction is required for efficient ribosome binding and histone acetylation. Furthermore, we provide insights on the potential coordination of methionine removal and subsequent NAA40-mediated acetylation by formation of a multienzyme complex on the ribosome involving METAP1. Therefore, our results illustrate the details of N-terminal histone acetylation by NAA40 and highlight the role of NAC as a general coordinator of nascent protein modification.

Background: N-terminal acetylation (Nt-Ac), mediated by N-terminal acetyltransferases (NATs) is one of the most abundant protein modifications occurring approximately in 80% of all eukaryotic proteins. In contrast to the broad spectrum NATs, the human N-alpha-acetyltransferase 40 (NAA40) is highly specific, currently known to Nt-acetylate only the two histone proteins H4 and H2A, which share an Ser(1)-Gly(2)-Arg(3)-Gly(4) N-terminal sequence. Previous work from our lab and others has highlighted the biological and clinical relevance of this NAA40-mediated modification. Results: In this study, by performing in silico analysis of protein sequences combined with biochemical assays we identify the histone variants H2A.X and H2A.J and the chromatin remodeler SMARCD2 as new potential substrates of human NAA40. Subsequently, focusing on H2A.X, we show for the first time by mass spectrometry analysis that H2A.X is N-terminally acetylated (Nt-acH2A.X) within human cells. Next, we demonstrate that NAA40 specifically interacts and N-terminally acetylates histone H2A.X, in vitro and within cells. Finally, we provide evidence that H2A.X N-terminal acetylation is responsive to Ultraviolet B (UVB)-induced DNA damage and its associated enzyme NAA40 affects the survival of cells exposed to UVB irradiation. Conclusion: Our findings identify H2A.X as a novel bona fide substrate of NAA40. Moreover, the responsiveness of H2A.X N-terminal acetylation to UV-induced DNA damage indicates that this is a dynamic modification with potential biological functions.

A critical aspect of Molecular Communications (MC) is the implementation of signal detection policies amidst noise. To date, noise characterizations within the MC field have predominantly drawn from methodologies found in wireless communications literature. In this study, we diverge from existing MC research by utilizing a newly developed experimental platform that employs yeast, allowing us to consider more realistic noise characterizations based on the relevant signaling pathways. We propose suitable signal detection mechanisms tailored to this experimental setup, which focuses on yeast cell-to-cell communications. Our analysis identifies gene transcription as the primary source of noise, and we utilize a Markov birth-death process model with Poisson arrivals and departures to characterize it. The noisy expression of the FUS1 gene is best represented using a mixed Gaussian distribution model. This model serves as a foundation for evaluating the performance of Maximum Likelihood Detection mechanisms in terms of Bit Error Rate (BER) for both symbol-by-symbol and sequence transmission schemes. Error analysis indicates that appropriate adjustments to the signal threshold can reduce errors to as low as 10%, which is not negligible. In contrast, the detection of symbol sequences demonstrates enhanced error performance, achieving error rates as low as 0.4%, albeit at the cost of increased computational complexity.

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity.

N-alpha-acetyltransferase 40 (NAA40) is an evolutionarily conserved N-terminal acetyltransferase (NAT) linked to oncogenesis and chemoresistance. A recent study reported the generation of a second, shorter NAA40 isoform (NAA40^S) through alternative translation, which we proceeded to further characterise. Notably, recombinant NAA40^S had a greater in vitro enzymatic activity and affinity towards its histone H2A/H4 substrates compared to full-length NAA40 (NAA40^L). Within cells, NAA40^S was enzymatically active, based on its ability to suppress the H2A/H4S1Ph antagonistic mark in CRISPR-generated NAA40 knockout cells. Finally, we show that in addition to alternative translation, the NAA40^S isoform could be derived from a primate and testis-specific transcript, which may align with the “out-of-testis” origin of recently evolved genes and isoforms. To summarise, our data reveal an even greater functional divergence between the two NAA40 isoforms than had been previously recognised.