Biology subjects

Wierzbicki, A.

Publications and source records attributed to Wierzbicki, A..

4 recordsLinked to original sources

bioRxiv2026

PPM1B utilizes a trinuclear metal architecture for phosphatase activity

The metal-dependent protein phosphatase (PPM/PP2C) family regulates innate immune and cell death pathways through reversible phosphorylation. Although these enzymes contain a conserved third Mg2+/Mn2+ ion (M3) that is essential for activity, its chemical role in phosphate hydrolysis has remained unclear. Here, we report studies that reveal PPM1B promotes cell death during Pseudomonas aeruginosa infection and utilizes a trinuclear metal center in which M3 directly coordinates the substrate phosphate, positioning it for in-line SN2 hydrolysis. In addition to substrate orientation, M3 positions a water molecule to protonate the departing alkoxide, stabilizing the leaving-group. Functionally, M3 substitutes for the arginine clamp in phosphoprotein phosphatases (PPP), revealing that these evolutionarily distinct phosphatase families have converged on the same chemical strategy through fundamentally different catalytic architectures. Together, these findings define a three-metal mechanism in PPM phosphatases and identify the M3 site as a rare and potentially druggable feature for immune and infectious diseases.

biochemistry↗

bioRxiv2026

Suppression of ITPK1 and IPMK activities impairs mTORC1 signaling in pancreatic β-cells and implicates IP5 in stabilizing activated mTORC1

mTORC1 integrates growth factor and nutrient signals to regulate cellular metabolism, yet there are no metabolites known to directly regulate mTORC1 activity in cells. Cryo-EM studies revealed that inositol hexakisphosphate (IP6) associates with the FAT domain of mTOR, suggesting that inositol phosphates may directly modulate mTOR activity. We previously showed that higher-order inositol phosphates enhance mTORC1 kinase activity and stability in vitro. Here, we investigated whether inositol phosphate metabolism regulates mTORC1 signaling in pancreatic {beta}-cells. Suppression or acute inhibition of inositol phosphate multikinase (IPMK), as well as knockdown of inositol trisphosphate kinase 1 (ITPK1), selectively reduced cellular IP5 levels without altering IP6 and resulted in impaired basal and insulin-stimulated mTORC1 signaling, particularly under physiological glucose and low growth factor conditions. Combined inhibition of IPMK and ITPK1 nearly abolished IP5 and reduced IP6, demonstrating that these enzymes compensate to supply IP5 for IP6 synthesis. Importantly, depletion of IP5 did not impair PI3K/Akt activation but accelerated termination of the mTORC1 signal, indicating a role for IP5 in stabilizing the active mTORC1 complex. Reduction of inositol phosphate levels did not prevent insulin- or glucose-induced mTORC1 activation, revealing that IP5 primarily regulates signal persistence rather than initiation. Together, these findings identify IP5 as a metabolic regulator that prolong mTORC1 activity in {beta}-cells, providing a mechanism by which cellular metabolic state modulates sustained mTORC1 signaling. Significance StatementmTORC1 is a central metabolic regulator whose chronic activation contributes to metabolic disease, yet mechanisms that sustain mTORC1 activity after its activation are poorly understood. We show that enzymes controlling inositol phosphate metabolism regulate the stability of mTORC1 signaling in pancreatic {beta}-cells by maintaining cellular levels of inositol pentakisphosphate (IP5). Reducing IP5 impairs basal and sustained mTORC1 signaling without affecting upstream growth factor or energy-sensing pathways, revealing a mechanism that controls signal duration rather than activation. These findings identify IP5 as a metabolic regulator of mTORC1 and suggest that targeting inositol phosphate metabolism may provide a strategy to modulate mTORC1 activity in metabolic disease.

biochemistry↗

bioRxiv2024

pTAC3 and pTAC14 are required for binding of plastid-encoded RNA polymerase to DNA

Plastid-encoded RNA polymerase (PEP) is a bacterial-type multisubunit RNA polymerase responsible for the majority of transcription in chloroplasts. PEP consists of four core subunits, which are orthologs of their cyanobacterial counterparts. In Arabidopsis thaliana, PEP associates with 12 PEP-associated proteins (PAPs), which serve as peripheral subunits of the RNA polymerase. The exact contributions of PAPs to PEP function are still poorly understood. We use ptChIP-seq to show that PAP1/pTAC3, a peripheral subunit of PEP, binds to the same genomic loci as RpoB, a core subunit of PEP. The pap1/ptac3 mutant shows a complete loss of RpoB binding to DNA throughout the genome, indicating that PAP1/pTAC3 is necessary for RpoB binding to DNA. A similar loss of RpoB binding to DNA is observed in the pap7/ptac14 mutant, which is defective in another peripheral PEP subunit. We propose that the peripheral subunits of PEP are required for the recruitment of core PEP subunits to DNA. KEY MESSAGEThe peripheral subunits of plastid-encoded RNA polymerase play a crucial role in recruiting the core PEP subunits to DNA in Arabidopsis chloroplasts.

plant biology↗

bioRxiv2022

High-resolution map of plastid encoded polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

Plastids are endosymbiotic organelles containing their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial-type, multi-subunit polymerase encoded by the plastid genome. The plastid encoded RNA polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome-wide scale at high resolution. We established a highly specific approach to detect the genome-wide pattern of PEP binding to chloroplasts DNA using ptChIP-seq. We found that in mature Arabidopsis thaliana chloroplasts, PEP has a complex DNA binding pattern with preferential association at genes encoding rRNA, tRNA, and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with the levels of RNA accumulation, which allowed estimating the quantitative contribution of transcription to RNA accumulation.

plant biology↗