Inflammatory caspase substrate specificities.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

The tetrapeptide sequence of IL-18 and IL-1ß regulates their recruitment and activation by inflammatory caspases.

Inflammasomes are multiprotein signaling complexes that activate the innate immune system. Canonical inflammasomes recruit and activate caspase-1, which then cleaves and activates IL-1ß and IL-18, as well as gasdermin D (GSDMD) to induce pyroptosis. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are known to cleave GSDMD, but their role in direct processing of other substrates besides GSDMD has remained unknown. Here, we show that CASP4/5 but not CASP11 can directly cleave and activate IL-18. However, CASP4/5/11 can all cleave IL-1ß to generate a 27-kDa fragment that deactivates IL-1ß signaling. Mechanistically, we demonstrate that the sequence identity of the tetrapeptide sequence adjacent to the caspase cleavage site regulates IL-18 and IL-1ß recruitment and activation. Altogether, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation that may aid in developing new therapeutics for immune-related disorders.

The tetrapeptide sequence of IL-1ß regulates its recruitment and activation by inflammatory caspases.

The mammalian innate immune system uses germline-encoded cytosolic pattern-recognition receptors (PRRs) to detect intracellular danger signals. At least six of these PRRs are known to form multiprotein complexes called inflammasomes which activate cysteine proteases known as caspases. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines such as IL-1ß and IL-18, as well as the pore forming protein, gasdermin D (GSDMD), to induce pyroptotic cell death. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are activated by intracellular LPS to cleave GSDMD, but their role in direct processing of inflammatory cytokines has not been established. Here we show that active CASP4/5 directly cleave IL-18 to generate the active species. Surprisingly, we also discovered that CASP4/5/11 cleave IL-1ß at D27 to generate a 27 kDa fragment that is predicted to be inactive and cannot signal to the IL-1 receptor. Mechanistically, we discovered that the sequence identity of the P4-P1 tetrapeptide sequence adjacent to the caspase cleavage site (D116) regulates the recruitment and processing of IL-1ß by inflammatory caspases to generate the bioactive species. Thus, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation.

De novo protein design of photochemical reaction centers.

Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here, we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.

First principles design of a core bioenergetic transmembrane electron-transfer protein.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Elementary tetrahelical protein design for diverse oxidoreductase functions.

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.

Engineering oxidoreductases: maquette proteins designed from scratch.

The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.

Biomimetic chemical sensors using nanoelectronic readout of olfactory receptor proteins.

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.

Direct probe of molecular polarization in de novo protein-electrode interfaces.

A novel approach to energy harvesting and biosensing devices would exploit optoelectronic processes found in proteins that occur in nature. However, in order to design such systems, the proteins need to be attached to electrodes and the optoelectronic properties in nonliquid (ambient) environments must be understood at a fundamental level. Here we report the simultaneous detection of electron transport and the effect of optical absorption on dielectric polarizability in oriented peptide single molecular layers. This characterization requires a peptide design strategy to control protein/electrode interface interactions, to allow peptide patterning on a substrate, and to induce optical activity. In addition, a new method to probe electronic, dielectric, and optical properties at the single molecular layer level is demonstrated. The combination enables a quantitative comparison of the change in polarization volume between the ground state and excited state in a single molecular layer in a manner that allows spatial mapping relevant to ultimate device design.

Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely hydrophobic cofactors to lipophilic domains.

We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.

Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.

We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

Amphiphilic four-helix bundle peptides designed for light-induced electron transfer across a soft interface.

A family of four-helix bundle peptides were designed to be amphiphilic, possessing distinct hydrophilic and hydrophobic domains along the length of the bundle's exterior. This facilitates their vectorial insertion across a soft interface between polar and nonpolar media. Their design also now provides for selective incorporation of electron donor and acceptor cofactors within each domain. This allows translation of the designed intramolecular electron transfer along the bundle axis into a macroscopic charge separation across the interface.

Molecular extensibility of mini-dystrophins and a dystrophin rod construct.

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.

Amphiphilic 4-helix bundles designed for biomolecular materials applications.

Artificial peptides previously designed to possess alpha-helical bundle motifs have been either hydrophilic (i.e., soluble in polar media) or lipophilic (i.e., soluble in nonpolar media) in overall character. Realizations of these bioinspired bundles have succeeded in reproducing a variety of biomimetic functionality within the appropriate media. However, to translate their functionality into any biomolecular device applications at the macroscopic level, the bundles must be oriented in an ensemble, for example, at an interface. This goal has been realized in a new family of alpha-helical bundle peptides which are amphiphilic; namely, they assemble into 4-helix bundles with well-defined hydrophilic and hydrophobic domains. These peptides are capable of binding metalloporphyrin prosthetic groups at selected locations within these domains. We describe here the realization of one of the first members of this family, AP0, successfully designed for vectorial incorporation into soft interfaces between polar and nonpolar media.

Functionalizing nanocrystalline metal oxide electrodes with robust synthetic redox proteins.

De novo designed synthetic redox proteins (maquettes) are structurally simpler, working counterparts of natural redox proteins. The robustness and adaptability of the maquette protein scaffold are ideal for functionalizing electrodes. A positive amino acid patch has been designed into a maquette surface for strong electrostatic anchoring to the negatively charged surfaces of nanocrystalline, mesoporous TiO(2) and SnO(2) films. Such mesoporous metal oxide electrodes offer a major advantage over conventional planar gold electrodes by facilitating formation of high optical density, spectroelectrochemically active thin films with protein loading orders of magnitude greater (up to 8 nmol cm(-2)) than that achieved with gold electrodes. The films are stable for weeks, essentially all immobilized-protein display rapid, reversible electrochemistry. Furthermore, carbon monoxide ligand binding to the reduced heme group of the protein is maintained, can be sensed optically and reversed electrochemically. Pulsed UV excitation of the metal oxide results in microsecond or faster photoreduction of an immobilized cytochrome and millisecond reoxidation. Upon substitution of the heme-group Fe by Zn, the light-activated maquette injects electrons from the singlet excited state of the Zn protoporphyrin IX into the metal oxide conduction band. The kinetics of cytochrome/metal oxide interfacial electron transfer obtained from the electrochemical and photochemical data obtained are discussed in terms of the free energies of the observed reactions and the electronic coupling between the protein heme group and the metal oxide surface.

Hydrophilic to amphiphilic design in redox protein maquettes.

De novo protein design has created small, robust protein-cofactor complexes that serve as simplified working models, or maquettes, for the much more complicated natural oxidoreductases. We review the research avenues that spring from the better characterized water-soluble hydrophilic maquettes and guide the construction of amphiphilic maquettes patterned on membrane-bound oxidoreductases that couple electron transfer to transmembrane proton-motive force. We address the special working challenges and opportunities that arise with amphiphilic maquettes designed to assemble in membranes, along with the redox and pigment cofactors required to promote light activated electron transfer, redox-coupled electric field generation, proton exchange and transmembrane charge motion.

Liquid-crystalline collapse of pulmonary surfactant monolayers.

During exhalation, the surfactant film of lipids and proteins that coats the alveoli in the lung is compressed to high surface pressures, and can remain metastable for prolonged periods at pressures approaching 70 mN/m. Monolayers of calf lung surfactant extract (CLSE), however, collapse in vitro, during an initial compression at approximately 45 mN/m. To gain information on the source of this discrepancy, we investigated how monolayers of CLSE collapse from the interface. Observations with fluorescence, Brewster angle, and light scattering microscopies show that monolayers containing CLSE, CLSE-cholesterol (20%), or binary mixtures of dipalmitoyl phosphatidylcholine(DPPC)-dihydrocholesterol all form bilayer disks that reside above the monolayer. Upon compression and expansion, lipids flow continuously from the monolayer into the disks, and vice versa. In several respects, the mode of collapse resembles the behavior of other amphiphiles that form smectic liquid-crystal phases. These findings suggest that components of surfactent films must collapse collectively rather than being squeezed out individually.

Effect of neutral lipids on coexisting phases in monolayers of pulmonary surfactant.

We previously established that compression of monolayers containing the lipids in pulmonary surfactant, with or without the surfactant proteins, initially leads to phase separation. On further compression, however, phase coexistence terminates at a critical point that requires the presence of cholesterol. The studies reported here address the changes in the phospholipid phase diagram produced by cholesterol. We used the two systems of the lipids from calf surfactant with and without the surfactant proteins. For both systems, we began with the postulate that cholesterol had no effect on the composition of other constituents in the two phases, and then used the known behavior of interfacial tension at a critical point to test the two extreme cases in which the cholesterol partitions exclusively into condensed domains or into the surrounding film. Measurements of surface potential along with the fraction of the nonfluorescent area and the radius of the domains, both obtained by fluorescence microscopy, for films with and without cholesterol allowed calculation of the interfacial tension between the two phases. Only the model that assumes the presence of cholesterol within the domains accurately predicts a decreasing line tension during film compression toward the critical point. That model, however, also predicts an unlikely decrease during compression of the dipole moment density for the condensed phase. Our results are best explained in terms of cholesterol partitioning predominantly into the condensed domains, with a resulting partial redistribution of the phospholipids between the two phases.