Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.

Immune cells activate in binary, switch-like fashion via large protein assemblies known as signalosomes, but the molecular mechanism of the switch is not yet understood. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, which governs NF-κB activation in both innate and adaptive immunity. We found that the switch consists of a sequence-encoded and deeply conserved nucleation barrier to ordered polymerization by the adaptor protein BCL10. The particular structure of the BCL10 polymers did not matter for activity. Using optogenetic tools and single-cell transcriptional reporters, we discovered that endogenous BCL10 is functionally supersaturated even in unstimulated human cells, and this results in a predetermined response to stimulation upon nucleation by activated CARD multimers. Our findings may inform on the progressive nature of age-associated inflammation, and suggest that signalosome structure has evolved via selection for kinetic rather than equilibrium properties of the proteins.

Innate immune responses, such as cell death and inflammatory signaling, are typically switch-like in nature. They also involve ''prion-like'' self-templating polymerization of one or more signaling proteins into massive macromolecular assemblies known as signalosomes. Despite the wealth of atomic-resolution structural information on signalosomes, how the constituent polymers nucleate and whether the switch-like nature of that event at the molecular scale relates to the digital nature of innate immune signaling at the cellular scale remains unknown. In this perspective, we review current knowledge of innate immune signalosome assembly, with an emphasis on structural constraints that allow the proteins to accumulate in inactive soluble forms poised for abrupt polymerization. We propose that structurally encoded nucleation barriers to protein polymerization kinetically regulate the corresponding pathways, which allows for extremely sensitive, rapid, and decisive signaling upon pathogen detection. We discuss how nucleation barriers satisfy the rigorous on-demand functions of the innate immune system but also predispose the system to precocious activation that may contribute to progressive age-associated inflammation.

Protein phase transitions broadly govern protein function and dysfunction. However, analyzing the consequences of specific phase transitions in cells is hindered by the low throughput and limited resolution of fluorescence microscopy, and this problem is compounded for proteins with complex phase behavior such as those implicated in age-associated neurodegenerative diseases. As one solution to this problem, we incorporated an orthogonally fluorescent proxy of total protein expression to adjust for effective cell volume differences in a flow cytometric assay for protein self-association Distributed Amphifluoric FRET (DAmFRET), thereby allowing the intracellular saturating concentrations of different proteins to be precisely compared in single experiments. We further found that the effective cell volume decreased in cells experiencing proteotoxicity, which provided a simple way to assign toxicity to specific phases of ectopically expressed proteins.

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Protein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed D istributed Am phifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies, and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior, and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.

Study ObjectiveTo identify and characterize the mechanism protein phosphatases use to regulate KCCs activity during ionic homeostasis and the regulation of cell volume via WNK3 kinase.BackgroundSLC12A family are membrane proteins that transport Na+ and/or K+ coupled to Clinside and outside the cell. They regulate cell volume, trans‐epithelial ionic transport, neuron excitability and blood pressure balance. Several members of the family and their regulatory proteins are mutated in human diseases characterized by alteration of ionic homeostasis mainly in the kidney and the brain. The family is formed by KCCs, NCC and NKCCs regulated in a reciprocal manner by phospho‐ and dephosphorylation events. WNKs kinases and their down‐stream substrates, SPAK/OSR1 are their main phospho‐regulatory proteins. Dephosphorylation results in KCCs activation and NCC and NKCCs inhibition, whereas phosphorylation produces the opposite effect. KCCs activation is abolished by inhibition of PP1A and PP2, demonstrating an essential regulatory role for PPs in this process. Substrate specificity for WNK and SPAK/OSR1 kinases is stablished by a SPAK binding motif, present in WNK kinases and the SLC12A. Conversely, some consensus motifs for direct PPs‐dephosphorylation have been stablished. In the present study we analyzed two putative PP1 binding sites present in the WNK3 kinase sequence. Our previous findings showed that WNK3 inhibits all the KCCs and its catalytically inactive form, WNK3 DA activates them. Inhibition of PPs showed regulation of KCCs activity by WNK3 depends on PPs activity. By mutating these putative PP1 binding sites we are aiming to identify the serine (Ser)‐threonine (Thr) kinases/phosphatases complex involved in KCCs response to cell volume changes regulated by WNK3.MethodsKCCs dephosphorylation was evaluated by Western Blot analysis. from HEK 293 cells transfected with either WT WNK3 kinase (WNK3 WT), catalytically inactive WNK3 (WNK3 DA), or PP1 binding sites mutant WNK3 (WNK3 PP1A, WNK3 DA PP1A, WNK3 PP1B or WNK3 DA PP1B) Cells were stimulated with either isotonic or hypotonic buffers in order to stimulate phosphatase activity under KCCs regulatory phosphorylation sites (pT1039 and pT991 in KCC3a). Regulation of KCCs K‐Cl transport by WNK3 WT, DA and mutants was assayed directly in Xenopus laevis oocytes. Formation of a kinase/phosphatase/cotransporter complex was evaluated by immunoprecipitation assays.ResultsData obtained in HEK cells and X. laevis oocytes showed that WNK3 PP1 binding sites mutation modified the effect of WNK3 WT and WNK3 DA previously reported over KCCs in response to cell volume changes.ConclusionsBinding of PP1 to WNK3 and formation of a regulatory complex is necessary for WNK3 to regulate SLC12A family response to osmotic and cell volume changes.Support or Funding InformationPAPIIT IA207718CONACYT, CB‐2016‐01, 283555This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.