The extracellular matrix (ECM) is a protein polymer network that physically supports cells within a tissue. It acts as an important physical and biochemical stimulus directing cell behaviors. For fibronectin (Fn), a predominant component of the ECM, these physical and biochemical activities are inextricably linked as physical forces trigger conformational changes that impact its biochemical activity. Here, we analyze whether oxidative post-translational modifications, specifically glutathionylation, alter Fn’s mechano-chemical characteristics through stretch-dependent protein modification. ECM post-translational modifications represent a potential for time- or stimulus-dependent changes in ECM structure-function relationships that could persist over time with potentially significant impacts on cell and tissue behaviors. In this study, we show evidence that glutathionylation of Fn ECM fibers is stretch-dependent and alters Fn fiber mechanical properties with implications on the selectivity of engaging integrin receptors. These data demonstrate the existence of multimodal post-translational modification mechanisms within the ECM with high relevance to the microenvironmental regulation of downstream cell behaviors. Post-translational modifications potentially alter the biochemical and biophysical properties of the extracellular matrix in significant ways. Here, the authors discover that glutathionylation alters the properties of the matrix protein fibronectin.

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLF WT ) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLF D380A ) and severe (OLF I499F ) disease variants aggregate differently, with rates comparable to OLF WT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLF WT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gβγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gβγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gβγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gβγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gβγ signaling axes.

Emerging evidence suggests that heterotrimeric G protein gamma subunits (Gγ) are important governors of G protein signaling, a function that is mediated through GPCR- and pH-dependent combinatorial phosphorylation of their intrinsically disordered N-terminal tails (Gγ-Nt) that controls Gβγ/effector interactions and signaling. Intrinsic disorder is a universally conserved structural feature of all Gγ subunit N-termini, which prompted us to hypothesize that, beyond phosphorylation, intrinsic disorder itself is inherently important to the signal-governing roles of Gγ subunits. To test this hypothesis we devised a strategy in which single amino acid substitutions are sequentially introduced into the Gγ tail, producing a series of isoforms that proceed step-wise from a fully-disordered to fully-ordered (α-helical) Nt tail structure. As a control for the increasing mutation load, we compare these mutants to those in which the same number of amino acid substitutions are incorporated that do not alter the inherent structural disorder of the tail. These mutant isoforms were then structurally analyzed by circular dichroism (CD) in vitro, by molecular dynamics (MD) simulation in silico, and by functional analysis of Gβγ-dependent molecular signaling in vivo. Here, we apply this approach to the yeast Gγ subunit, Ste18. CD and MD analyses of Ste18-Nt tail isoforms indicate that a successful transition from a fully-disordered to fully-ordered state is achievable through precise point mutation. Replacing the wild type Gγ subunit with each of the mutant isoforms in yeast, we further show that intrinsic disorder of Gγ-Nt controls the stability of the Gγ subunit in a manner that is proportional to the loss of intrinsic disorder in vivo. pH-dependent phosphorylation at Ser3 in the tail is largely unaffected by these changes. However, unexpectedly, we found that the GPCR-dependent phosphorylation site, Ser7, becomes pH-sensitive in response to changes in tail structure. Ongoing experiments reveal the effects of Ste18-Nt tail structure on the interaction of yeast Gbg with its primary effector Ste5, and subsequent effects on activation of MAPKs, which have been shown to be highly sensitive to Gγ-Nt tail phosphorylation previously. Taken together, these data provide evidence that intrinsic structural disorder plays a direct role in functionality of Gγ subunits as governors of G proteins signaling and substantiates the rationale for exploring similar roles for these tails in mammalian Gβγ-dependent signaling pathways.

Protein intrinsically disordered regions (IDRs) are often targets of combinatorial post-translational modifications (PTMs) that serve to regulate protein structure and/or function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits – essential components of heterotrimeric G protein complexes – are intrinsically disordered, highly phosphorylated governors of G protein signaling. Here, we demonstrate that the yeast Gγ Ste18 undergoes combinatorial, multi-site phosphorylation within its N-terminal IDR. Phosphorylation at S7 is responsive to GPCR activation and osmotic stress while phosphorylation at S3 is responsive to glucose stress and is a quantitative indicator of intracellular pH. Each site is phosphorylated by a distinct set of kinases and both are also interactive, such that phosphomimicry at one site affects phosphorylation on the other. Lastly, we show that phosphorylation produces subtle yet clear changes in IDR structure and that different combinations of phosphorylation modulate the activation rate and amplitude of the scaffolded MAPK Fus3. These data place Gγ subunits among the growing list of intrinsically disordered proteins that exploit combinatorial post-translational modification to govern signaling pathway output.

Intracellular heterotrimeric G proteins (consisting of Gα, Gβ, and Gγ subunits), interface with 7‐transmembrane G protein coupled receptors (GPCRs) and function as the primary transducers of extracellular signals such as hormones, neurotransmitters, as well as pharmaceutical compounds. Upon binding their cognate ligands, GPCRs promote the dissociation of Gα and Gβγ subunits, which are then free to interact with effector proteins that promote intracellular signaling responses to the stimulus. Within this context, Gγ subunits are thought of primarily as membrane anchors for Gβ subunits, which must reside at the plasma membrane to facilitate functional protein interactions. However, emerging evidence suggests that Gγ subunits serve as phosphorylation‐dependent governors of G protein signaling. We have recently demonstrated this in the budding yeast model system, in which negative feedback phosphorylation of the intrinsically disordered N‐terminal tail of Gγ/Ste18 disrupts Gβγ/effector interactions and inhibits the normal GPCR‐dependent activation of MAPKs (Choudhury S. et al, Cell Rep. 2018). More recently, we have discovered that the yeast Gγ subunit undergoes combinatorial phosphorylation in response to several different forms of extracellular stimuli, including osmotic stress, pH stress, glucose deprivation, and cell cycle progression, in addition to GPCR activation. In light of these discoveries, we have hypothesized that Gγ phosphorylation plays a similar role in mammalian GPCR pathways. Indeed, we show that, like Gγ/Ste18, all mammalian Gγ subunits have an intrinsically disordered N‐terminus and most also undergo multi‐site phosphorylation. Here we provide new evidence to evaluate the hypothesis that Gγ phosphorylation is a governor of G protein signaling in mammals by demonstrating that phosphorylation is readily detectable on many different human Gγ subunits (Gγ3, 4, 5, 7, 10, 12) and by systematically testing the requirement for phosphorylation in proper Gβγ‐specific signaling pathways including the activation of PKD1 kinase by phospholipase C β2/3.Support or Funding InformationNIH