Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts.

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel ß subunit (ß1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.

Correlated STORM-homoFRET imaging reveals highly heterogeneous membrane receptor structures.

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3ß.

Glycogen synthase kinase 3ß (GSK3ß) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3ß substrates, such as c-Myc and Snail, are nuclear transcription factors, suggesting the possibility that GSK3ß function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3ß from the cytosol to the nucleus and to a GSK3ß-dependent reduction of the levels of both c-Myc and Snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3ß nuclear localization in an mTORC1-dependent manner. GSK3ß was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through a perturbation of RAS oncogene family member 7 (Rab7) resulted in loss of GSK3ß from lysosomes and in enhanced GSK3ß nuclear localization as well as GSK3ß-dependent reduction of c-Myc levels. These findings indicate that the nuclear localization and function of GSK3ß is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3ß-dependent regulation of transcriptional networks controlling cellular biomass production.

Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.

Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking.

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.

Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a ß-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation.

A lateral signalling pathway coordinates shape volatility during cell migration.

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1-Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration.

The mechanism of membrane disruption by cytotoxic amyloid oligomers formed by prion protein(106-126) is dependent on bilayer composition.

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106-126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106-126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Forces of interactions between iron and aluminum silicates: effect of water chemistry and polymer coatings.

Atomic force microscopy-based force spectroscopy (AFM) was employed to investigate the forces of interaction between aluminum silicates (mica and a synthetic aluminum-silicate) and iron particles, both bare and coated with carboxymethyl cellulose (CMC) polymer. Experiments were conducted in water and salt solutions (100mM NaCl and 100mM CaCl2) at pH 5.5, in water at pH 4 and 8, and in 10mg/l humic acid solutions. In addition, humic acid sorption onto the synthetic aluminum-silicate was probed with a quartz crystal microbalance with dissipation monitoring (QCM-D). Interactions between bare iron particles and aluminum silicate were attractive except at pH 8 and in the presence of humic acids in which case forces upon approach were repulsive. Interactions between bare iron and mica were similar, except that repulsive forces upon approach were measured in 100mM NaCl solutions, possibly due to increased hydration of mica compared to aluminum silicate. Interactions between CMC coated iron particles and aluminum-silicates were either repulsive or at most weakly attractive, likely due to repulsive electro-steric forces associated with the CMC. QCM-D results indicated that humic acids adsorbed to aluminum silicate, producing electro-steric repulsion to coated and uncoated iron. AFM data were successfully modeled using extended DLVO theory and a modified Ohshima's model. This modeling provided insights into the contributions of various processes to the measured interaction forces, highlighting the importance of van der Waals and hydration forces.

Inside-out signaling promotes dynamic changes in the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) oligomeric state to control its cell adhesion properties.

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded (432)GXXXG(436) motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the (432)GXXXG(436) motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the (432)GXXXG(436) mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.

Forces of interactions between bare and polymer-coated iron and silica: effect of pH, ionic strength, and humic acids.

The interactions between a silica substrate and iron particles were investigated using atomic force microscopy-based force spectroscopy (AFM). The micrometer- and nanosized iron particles employed were either bare or coated with carboxymethyl cellulose (CMC), a polymer utilized to stabilize iron particle suspensions. The effect of water chemistry on the forces of interaction was probed by varying ionic strength (with 100 mM NaCl and 100 mM CaCl2) or pH (4, 5.5, and 8) or by introducing 10 mg/L of humic acids (HA). When particles were uncoated, the forces upon approach between silica and iron were attractive at pH 4 and 5.5 and in 100 mM CaCl2 at pH 8, but they were negligible in 100 mM NaCl buffered to pH 8 and repulsive in water buffered to pH 8 and in HA solutions. HA produced electrosteric repulsion between iron particles and silica, likely due to its sorption to iron particles. HA sorption to silica was excluded on the basis of experiments conducted with a quartz-crystal microbalance with dissipation monitoring. Repulsion with CMC-coated iron was attributed to electrosteric forces, which were damped at high ionic strength. An extended DLVO model and a modified version of Ohshima's theory were successfully utilized to model AFM data.

Effect of water chemistry and aging on iron-mica interaction forces: implications for iron particle transport.

The transport of particles through groundwater systems is governed by a complex interplay of mechanical and chemical forces that are ultimately responsible for binding to geological substrates. To understand these forces in the context of zero valent iron particles used in the remediation of groundwater, atomic force microscopy (AFM)-based force spectroscopy was employed to characterize the interactions between AFM tips modified with either carbonyl iron particles (CIP) or electrodeposited Fe as a function of counterion valency, temperature, particle morphology, and age. The measured interaction forces were always attractive for both fresh and aged CIP and electrodeposited iron, except in 100 mM NaCl, as a consequence of electrostatic attraction between the negatively charged mica and positively charged iron. In 100 mM NaCl, repulsive hydration forces appeared to dominate. Good agreement was found between the experimental data and predictions based on the extended DLVO (XDLVO) theory. The effect of aging on iron particle composition and morphology was assessed by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) revealing that the aged particles comprising a zero valent iron core passivated by a mixture of iron oxides and hydroxides. Force spectroscopy showed that aging caused variations in the adhesive force due to the changes in particle morphology and contact area.

Nucleation and growth of elastin-like peptide fibril multilayers: an in situ atomic force microscopy study.

Controlling how molecules assemble into complex supramolecular architectures requires careful consideration of the subtle inter- and intra-molecular interactions that control their association. This is particularly crucial in the context of assembly at interfaces, where both surface chemistry and structure can play a role in directing structure formation. We report here the results of a study into the self-assembly of the elastin-like peptide EP I on structurally modified highly ordered pyrolytic graphite, including the role of spatial confinement on fibril nucleation and the growth of oriented fibril multilayers. In situ atomic force microscopy performed in fluid and at elevated temperature provided direct evidence of frustrated fibril nuclei and oriented growth of independent fibril domains. These results portend the application of this in situ strategy for studies of the nucleation and growth mechanisms of other fibril- and amyloid-forming proteins.

Dynamic macrophage "probing" is required for the efficient capture of phagocytic targets.

Binding of ligands by immunoreceptors is thought to be a passive, stochastic process. Contrary to this notion, we found that binding of IgG-opsonized particles by Fcγ receptors was inhibited in macrophages, dendritic and microglial cells by agents that interfere with actin assembly or disassembly. Changes in the lateral mobility of the receptors--assessed by single-particle tracking--or in the microelasticity of the membrane--determined by atomic-force microscopy--could not account for the effects of actin disruption on particle binding. Instead, we found that the macrophages contact their targets by actively extending actin-rich structures. Formation of these protrusions is driven by Rac and requires phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Capture of C3bi-opsonized as well as unopsonized targets by macrophages was also dependent on actin. Thus, phagocytes continuously probe their environment for foreign particles in a manner akin to the constitutive sampling of the fluid milieu by dendritic cells. Active probing by phagocytes is most important when confronted by scarcely opsonized and/or highly mobile targets.

Peptide-induced domain formation in supported lipid bilayers: direct evidence by combined atomic force and polarized total internal reflection fluorescence microscopy.

Direct visualization of the mechanism(s) by which peptides induce localized changes to the structure of membranes has high potential for enabling understanding of the structure-function relationship in antimicrobial and cell-penetrating peptides. We have applied a combined imaging strategy to track the interaction of a model antimicrobial peptide, PFWRIRIRR-amide, with bacterial membrane-mimetic supported phospholipid bilayers comprised of POPE/TOCL. Our in situ studies revealed rapid reorganization of the POPE/TOCL membrane into localized TOCL-rich domains with a concomitant change in the organization of the membranes themselves, as reflected by changes in fluorescent-membrane-probe order parameter, upon introduction of the peptide.

Biodegradable quantum dot nanocomposites enable live cell labeling and imaging of cytoplasmic targets.

Semiconductor quantum dots (QDs) offer great promise as the new generation of fluorescent probes to image and study biological processes. Despite their superior optical properties, QDs for live cell monitoring and tracking of cytoplasmic processes remain limited due to inefficient delivery methods available, altered state or function of cells during the delivery process and the requirement of surface-functionalized QDs for specific labeling of subcellular structures. Here, we present a noninvasive method to image subcellular structures in live cells using bioconjugated QD nanocomposites. By incorporating antibody-coated QDs within biodegradable polymeric nanospheres, we have designed a bioresponsive delivery system that undergoes endolysosomal to cytosolic translocation via pH-dependent reversal of nanocomposite surface charge polarity. Upon entering the cytosol, the polymer nanospheres undergo hydrolysis thus releasing the QD bioconjugates. This approach facilitates multiplexed labeling of subcellular structures inside live cells without the requirement of cell fixation or membrane permeabilization. As compared to conventional intracellular delivery techniques, this approach allows the high throughput cytoplasmic delivery of QDs with minimal toxicity to the cell. More importantly, this development demonstrates an important rational strategy for the design of a multifunctional nanosystem for biological applications.

Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation.

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

Reversible assembly of helical filaments by de novo designed minimalist peptides.

We have designed a series of 15 short, helical de novo peptides consisting of lysine, isoleucine, and alanine. We have termed this the KIA series. These peptides differ only in their hydrophobic interface, and thus their self-association is largely a consequence of hydrophobic interactions. One of these peptides, KIA13, forms insoluble helical fibers at specific NaCl concentrations. We have used CD spectroscopy, turbidity assays, and in situ tapping mode atomic force microscopy to characterize the reversible assembly pathway for this peptide. It is unfolded at low NaCl concentration, and forms helical, soluble fibers resembling a coiled-coil conformation at intermediate NaCl concentrations, and rope-like insoluble fibers at high NaCl concentrations. Reducing the NaCl concentration completely reverses this process. Another peptide from the KIA series specifically inhibits the formation of the insoluble KIA13 fibers, and reverses the process to some extent. This work sheds light onto protein fibrillogenesis and offers intriguing possibilities for the use of these types of peptides in drug delivery and biomaterials applications.