Biology subjects

Hageman, M.

Publications and source records attributed to Hageman, M..

3 recordsLinked to original sources

bioRxiv2025

Rapid optimization of protein function in mammalian cells via microbe-independent deep assembly and screening

Random mutagenesis and deep mutational scanning (DMS) are widely used to optimize proteins by oversampling large libraries in microbial cells, selecting cells expressing favorable variants, and sequencing to identify enriched variants. However, these methods are slow and costly, require effort to establish selection methods for a given protein function, and do not yield data on lower-performing variants. Here, we describe Microbe-Independent Deep Assembly and Screening (MIDAS), a rapid, high-throughput method for optimizing protein function directly in mammalian cells. As a demonstration, we applied MIDAS to improve a newly designed neurotransmitter bioluminescent indicator (NeuBI) for acetylcholine (ACh). MIDAS systematically optimized interdomain linkers, identified mutational hotspots, and exhaustively scanned amino acid combinations, in each case relating specific sequences to protein performance. MIDAS-optimized variants exhibited improved performance in vivo, highlighting the potential of MIDAS for improving protein function in mammalian systems.

bioengineering↗

bioRxiv2024

Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication

Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFN{gamma}, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness. IMPORTANCECoronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.

microbiology↗

bioRxiv2021

Phycobilisome breakdown effector NblD is required to maintain the cellular amino acid composition during nitrogen starvation

Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. ImportanceCyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.

microbiology↗