Biology subjects

Ustione, A.

Publications and source records attributed to Ustione, A..

6 recordsLinked to original sources

bioRxiv2026

Protonation- and substrate-regulated dimer opening couples brain-type creatine kinase to vesicular and actin-remodeling membranes

Brain-type creatine kinase (CK-BB) buffers local ATP demand through reversible phosphotransfer between ATP and phosphocreatine, yet how this soluble metabolic enzyme engages membrane compartments is unknown. Here, we combine fluorescence microscopy, DEER spectroscopy, hydrogen-deuterium exchange and native mass spectrometry, DEER- and AlphaFold-guided modeling, and long-timescale molecular dynamics to define the pH- and substrate-regulated conformational landscape governing CK-BB membrane association. Acidification promotes curvature-sensitive membrane binding and redistributes endogenous and recombinant CK-BB from diffuse cytosolic pools to punctate vesicular structures and membrane ruffles. Substrates independently promote curvature-sensitive association at neutral pH. DEER and modeling reveal an asymmetric dimer in which the convex surface remains restrained, whereas the concave catalytic-regulatory surface samples pH- and substrate-dependent intermediates. We identify progressive dimer opening as a novel regulatory mechanism whereby acidification and substrate binding increase dynamics across the convex surface and N-terminal dimer interface, generating membrane-competent conformations that facilitate curvature sensing and membrane association. Substrate binding buffers acid-induced deprotection while preserving dynamics near the His191/Ser199 regulatory interface. These findings establish CK-BB as a previously unrecognized curvature-sensitive metabolic enzyme and define dimer dynamics as a molecular switch coupling protonation and substrate occupancy to curved-membrane recognition and localized ATP regeneration, with potential relevance to neurodegeneration and cellular stress.

biophysics↗

bioRxiv2026

Circadian temperature compensation is intrinsically linked with metabolism and redox signaling

Circadian rhythms are a universal property of organisms, and a defining property of these oscillators is the ability to maintain a precise period at different temperatures. Indeed, the discovery in the 1950s of this property, known as "temperature compensation," was a watershed event in our understanding of biological timekeeping. Since that time, however, almost no general principles have emerged that uncover the mechanistic basis of how circadian timekeepers defy the rules" of temperature dependence of biochemical reactions. A genetic investigation of circadian temperature compensation revealed that the sensing of metabolism and ensuing modulation of the core mechanism preserves a constant circadian period. Intracellular redox sensing of metabolic rate is intrinsic to this compensation, and this relationship is conserved in bacterial and mammalian cells. These insights explain previously inexplicable observations of the interaction between redox reactions and circadian timekeeping. These results introduce a previously unknown linkage between temperature compensation and metabolism.

cell biology↗

bioRxiv2024

Insulin-Independent Regulation of Type 1 Diabetes via Brown Adipocyte-Secreted Proteins and the Novel Glucagon Regulator Nidogen-2.

Current treatments for type 1 diabetes (T1D) focus on insulin replacement. We demonstrate the therapeutic potential of a secreted protein fraction from embryonic brown adipose tissue (BAT), independent of insulin. The large molecular weight secreted fraction mediates insulin receptor-dependent recovery of euglycemia in a T1D animal model, nonobese diabetic (NOD) mice, by suppressing glucagon secretion. This fraction also promotes white adipocyte differentiation and browning, maintains healthy BAT, and enhances glucose uptake in adipose tissue, skeletal muscle, and liver. From this fraction, we identify nidogen-2 as a critical BAT-secreted protein that reverses hyperglycemia in NOD mice, inhibits glucagon secretion from pancreatic -cells, and mimics other actions of the entire secreted fraction. These findings confirm that BAT transplants affect physiology and demonstrate that BAT-secreted peptides represent a novel therapeutic approach to diabetes management. Furthermore, our research reveals a novel signaling role for nidogen-2, beyond its traditional classification as an extracellular matrix protein. HIGHLIGHTSO_LIThe large molecular weight brown adipocyte-secreted protein fraction suppresses glucagon secretion and normalizes glycemia in mouse models of type 1 diabetes (T1D), independent of insulin, offering a novel therapeutic strategy for disease management. C_LIO_LINidogen-2, a critical component of this fraction, is identified as an inhibitor of glucagon secretion in pancreatic -cells by regulating intracellular messenger activities. C_LIO_LIThe large-secreted protein fraction prevents T1D-related whitening of brown adipose tissue, promotes adipocyte differentiation, and enhances browning of inguinal white adipose tissue. C_LIO_LIThis fraction enhances glucose uptake in adipose tissue, skeletal muscle, and liver through an insulin receptor-dependent pathway. C_LI

cell biology↗

bioRxiv2021

SGLT2 inhibitors therapy protects glucotoxicity-induced β-cell failure in a mouse model of human KATP-induced diabetes trough mitigation of oxidative and ER stress

Progressive loss of pancreatic {beta}-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and {beta}-cell exhaustion. However, loss of {beta}-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the mechanisms underlying and temporal progression of glucotoxicity-induced loss of functional {beta}-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and proinsulin accumulation at 2-weeks of diabetes. This was accompanied by a marked increase in {beta}-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, early treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring {beta}-cell function in NDM mice when tit was initiated at >40 days of diabetes, when loss of {beta}-cell mass and identity had already occurred. These results have important clinical implications as they demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives {beta}-cell failure in diabetes, ii) recovery of {beta}-cell function by SGLT2 inhibitors is through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent {beta}-cell failure when used in early stages of diabetes, but not when loss of {beta}-cell mass/identity already occurred, iv) common execution pathways underlie loss and recovery of {beta}-cell function in different forms of diabetes.

cell biology↗

bioRxiv2019

Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins

Fluorescence fluctuation spectroscopy (FFS) can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis (SpIDA) and fluorescence moment image analysis (FMIA) are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color SpIDA (2c-SpIDA) and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering (SSF) technique to remove artifacts due to intrinsic crosstalk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species, hence this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with EGFP and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.\n\nStatement of SignificanceFluorescence fluctuation spectroscopy (FFS) techniques determine biophysical parameters from samples containing fluorescently labeled biomolecules by considering the statistical nature of fluorescent signals measured with photodetectors. The present study introduces two-color spatial cumulant analysis (2c-SpCA) to the canon of FFS techniques. 2c-SpCA analyzes pixel-value data of two-color images collected with laser scanning fluorescence microscopes. We show that 2c-SpCA can determine several biophysical parameters in living cells including Forster resonance energy transfer efficiency, the dark state fraction of fluorescent proteins, and heteromerization between distinctly labeled proteins. In comparison to existing techniques, 2c-SpCA requires very few image frames for analysis, minimal computations, and can be applied to images of fixed tissue samples.

biophysics↗

bioRxiv2017

FUCCI tracking shows that Neurog3 levels vary with cell-cycle phase in endocrine-biased pancreatic progenitors

Neurog3HI endocrine-committing cells are generated from a population of Sox9+ mitotic progenitors with only a low level of Neurog3 transcriptional activity (Neurog3TA.LO). Low-level Neurog3 protein, in Neurog3TA.LO cells, is required to maintain their mitotic endocrine-lineage-primed status. Herein, we describe a Neurog3-driven FUCCI cell-cycle reporter (Neurog3P2A.FUCCI) derived from a Neurog3 BAC transgenic reporter that functions as a loxed cassette acceptor (LCA). In cycling Sox9+ Neurog3TA.LO progenitors, the majority of cells in S-G2-M phases have undetectable levels of Neurog3 with increased expression of endocrine progenitor markers, while those in G1 have low Neurog3 levels with increased expression of endocrine differentiation markers. These findings support a model in which variations in Neurog3 protein levels are coordinated with cell-cycle phase progression in Neurog3TA.LO progenitors with entrance into G1 triggering a concerted effort, beyond increasing Neurog3 levels, to maintain an endocrine-lineage-primed state by initiating expression of the downstream endocrine differentiation program prior to endocrine-commitment.

cell biology↗