On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

Regulations of myo-inositol homeostasis: Mechanisms, implications, and perspectives.

Phosphorylation is the most common module of cellular signalling pathways. The dynamic nature of phosphorylation, which is conferred by the balancing acts of kinases and phosphatases, allows this modification to finely control crucial cellular events such as growth, differentiation, and cell cycle progression. Although most research to date has focussed on protein phosphorylation, non-protein phosphorylation substrates also play vital roles in signal transduction. The most well-established substrate of non-protein phosphorylation is inositol, whose phosphorylation generates many important signalling molecules such as the second messenger IP3, a key factor in calcium signalling. A fundamental question to our understanding of inositol phosphorylation is how the levels of cellular inositol are controlled. While the availability of protein phosphorylation substrates is known to be readily controlled at the levels of transcription, translation, and/or protein degradation, the regulatory mechanisms that control the uptake, synthesis, and removal of inositol are underexplored. Potentially, such mechanisms serve as an important layer of regulation of cellular signal transduction pathways. There are two ways in which mammalian cells acquire inositol. The historic use of radioactive 3H-myo-inositol revealed that inositol is promptly imported from the extracellular environment by three specific symporters SMIT1/2, and HMIT, coupling sodium or proton entry, respectively. Inositol can also be synthesized de novo from glucose-6P, thanks to the enzymatic activity of ISYNA1. Intriguingly, emerging evidence suggests that in mammalian cells, de novo myo-inositol synthesis occurs irrespective of inositol availability in the environment, prompting the question of whether the two sources of inositol go through independent metabolic pathways, thus serving distinct functions. Furthermore, the metabolic stability of myo-inositol, coupled with the uptake and endogenous synthesis, determines that there must be exit pathways to remove this extraordinary sugar from the cells to maintain its homeostasis. This essay aims to review our current knowledge of myo-inositol homeostatic metabolism, since they are critical to the signalling events played by its phosphorylated forms.

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase.

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition.

Functions for diverse metabolic activities in heterochromatin.

Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1's functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes.