Biology subjects

Mayfield, J. E.

Publications and source records attributed to Mayfield, J. E..

5 recordsLinked to original sources

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2024

Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription

The C-terminal domain of RPB1 (CTD) orchestrates transcription by recruiting regulators to RNA Pol II upon phosphorylation. Recent insights highlight the pivotal role of CTD in driving condensate formation on gene loci. Yet, the molecular mechanism behind how CTD-mediated recruitment of transcriptional regulators influences condensates formation remains unclear. Our study unveils that phosphorylation reversibly dissolves phase separation induced by the unphosphorylated CTD. Phosphorylated CTD, upon specific association with transcription regulatory proteins, forms distinct condensates from unphosphorylated CTD. Function studies demonstrate CTD variants with diverse condensation properties in vitro exhibit difference in promoter binding and mRNA co-processing in cells. Notably, varying CTD lengths lead to alternative splicing outcomes impacting cellular growth, linking the evolution of CTD variation/length with the complexity of splicing from yeast to human. These findings provide compelling evidence for a model wherein post-translational modification enables the transition of functionally specialized condensates, highlighting a co-evolution link between CTD condensation and splicing.

cell biology↗

bioRxiv2021

Ca2+-dependent liquid-liquid phase separation underlies intracellular Ca2+ stores.

Endoplasmic/sarcoplasmic reticulum Ca2+ stores are essential to myriad cellular processes, however, the structure of these stores is largely unknown and existing models do not address all literature observations. We investigate CASQ1 - the major Ca2+ binding protein of skeletal muscle - and discover Ca2+-dependent liquid-liquid phase separation activity. The intrinsic disorder of CASQ1 underlies this activity and is regulated via phosphorylation by the secretory pathway kinase FAM20C. This divalent cation driven condensation demonstrates liquid-liquid phase separation occurs within the endoplasmic/sarcoplasmic reticulum, mechanistically explains efficient Ca2+ buffering and storage, and represents a largely unexplored mechanism of divalent-cation driven protein association.

biochemistry↗

bioRxiv2020

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd {approx} 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide.

biochemistry↗

bioRxiv2019

Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase IIs C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTDs coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

biochemistry↗