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1.
J Am Chem Soc ; 146(5): 3250-3261, 2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38266489

RESUMO

Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stresses─unassisted by any protein activity─can induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.


Assuntos
Fosfolipídeos , Lipossomas Unilamelares , Pressão Osmótica , Lipossomas Unilamelares/metabolismo , Osmose , Água
2.
Langmuir ; 38(29): 8805-8816, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35816731

RESUMO

Lamellar mesophases of insoluble lipids are readily solubilized by the micellar mesophases of soluble surfactants. This simple process underscores a broad array of biochemical methodologies, including purification, reconstitution, and crystallization of membrane proteins, as well as the isolation of detergent-resistant membrane fractions. Although much is now known about the thermodynamic driving forces of membrane solubilization, the kinetic pathways by which the surfactant alters vesicular mesophases are only beginning to be appreciated. Little is known about how these interactions affect the solubilization of more complex, multilamellar mesophases. Here, we investigate how a common zwitterionic detergent affects the solubilization of a smectic, multilamellar, cylindrical mesophase of lipids, called the myelin figure. Our results reveal that myelin solubilization occurs in a multistep manner, producing a well-defined sequence of morphologically distinct intermediates en route to complete solubilization. The kinetic processes producing these intermediates include (1) coiling, which encompasses the formation, propagation, and tightening of extended helices; (2) thinning, which reflects the unbinding of lamellae in the smectic stacks; and (3) detachment or retraction, which either dissociates the myelinic protrusion from the source lipid mass or returns the myelinic protrusion to the source lipid mass─all in transit toward complete solubilization. These occasionally overlapping steps are most pronounced in single-lipid component myelins, while compositionally graded multicomponent myelins inhibit the coiling step and detach more frequently. Taken together, the appearance of these intermediates during the solubilization of myelins suggests a complex free-energy landscape characterizing myelin solubilization populated by metastable, morphological intermediates correlated with locally minimized changes in energy dependent upon the mesophase's composition. This then predicts the accessibility of structurally distinct, kinetic intermediates─such as loose and tight coiled helices, peeled myelins, retracted tubes, and detached protrusions─before reaching the stable ground state corresponding to a dissolved suspension of mixed surfactant-lipid micelles.


Assuntos
Surfactantes Pulmonares , Tensoativos , Detergentes/química , Excipientes , Lipídeos , Micelas , Bainha de Mielina , Solubilidade , Tensoativos/química
3.
Langmuir ; 38(3): 1045-1056, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35020400

RESUMO

When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figures─tens of micrometers wide and tens to hundreds of micrometers long─representing a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-ß-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration.


Assuntos
Bicamadas Lipídicas , Esfingomielinas , Fenômenos Biofísicos , Colesterol
4.
Mol Biol Evol ; 37(11): 3083-3093, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32521018

RESUMO

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.


Assuntos
Células Artificiais/química , Divisão Celular , Lactalbumina/química , Membranas/química , Ácido Oleico/química , Vesículas Citoplasmáticas/química , Peptídeos/química
5.
Biomacromolecules ; 20(4): 1709-1718, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30856330

RESUMO

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.


Assuntos
Hepacivirus/química , Membranas Artificiais , Peptídeos/química , Transição de Fase , Proteínas não Estruturais Virais/química , Domínios Proteicos
6.
Phys Chem Chem Phys ; 21(22): 11903-11915, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31125035

RESUMO

Biobutanol production by fermentation is potentially a sustainable alternative to butanol production from fossil fuels. However, the toxicity of butanol to fermentative bacteria, resulting largely from cell membrane fluidization, limits production titers and is a major factor limiting the uptake of the technology. Here, studies were undertaken, in vitro and in silico, on the butanol effects on a representative bacterial (i.e. Escherichia coli) inner cell membrane. A critical butanol : lipid ratio for stability of 2 : 1 was observed, computationally, consistent with complete interdigitation. However, at this ratio the bilayer was ∼20% thicker than for full interdigitation. Furthermore, butanol intercalation induced acyl chain bending and increased disorder, measured as a 27% lateral diffusivity increase experimentally in a supported lipid bilayer. There was also a monophasic Tm reduction in butanol-treated large unilamellar vesicles. Both behaviours are inconsistent with an interdigitated gel. Butanol thus causes only partial interdigitation at physiological temperatures, due to butanol accumulating at the phospholipid headgroups. Acyl tail disordering (i.e. splaying and bending) fills the subsequent voids. Finally, butanol short-circuits the bilayer and creates a coupled system where interdigitated and splayed phospholipids coexist. These findings will inform the design of strategies targeting bilayer stability for increasing biobutanol production titers.


Assuntos
1-Butanol/química , Membrana Celular/química , Bicamadas Lipídicas/química , Escherichia coli/química , Simulação de Dinâmica Molecular , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , Temperatura de Transição , Lipossomas Unilamelares/química
7.
Angew Chem Int Ed Engl ; 57(27): 8069-8072, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29707869

RESUMO

Membrane-intercalating conjugated oligoelectrolytes (COEs) are emerging as potential alternatives to conventional, yet increasingly ineffective, antibiotics. Three readily accessible COEs, belonging to an unreported series containing a stilbene core, namely D4, D6, and D8, were designed and synthesized so that the hydrophobicity increases with increasing side-chain length. Decreased aqueous solubility correlates with increased uptake by E. coli. The minimum inhibitory concentration (MIC) of D8 is 4 µg mL-1 against both E. coli and E. faecalis, with an effective uptake of 72 %. In contrast, the MIC value of the shortest COE, D4, is 128 µg mL-1 owing to the low cellular uptake of 3 %. These findings demonstrate the application of rational design to generate efficacious antimicrobial COEs that have potential as low-cost antimicrobial agents.


Assuntos
Anti-Infecciosos/química , Desenho de Fármacos , Polieletrólitos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Varredura Diferencial de Calorimetria , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Testes de Sensibilidade Microbiana , Polieletrólitos/síntese química , Polieletrólitos/farmacologia , Estilbenos/química
8.
Biophys J ; 112(8): 1682-1691, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28445759

RESUMO

The response of lipid bilayers to osmotic stress is an important part of cellular function. Recent experimental studies showed that when cell-sized giant unilamellar vesicles (GUVs) are exposed to hypotonic media, they respond to the osmotic assault by undergoing a cyclical sequence of swelling and bursting events, coupled to the membrane's compositional degrees of freedom. Here, we establish a fundamental and quantitative understanding of the essential pulsatile behavior of GUVs under hypotonic conditions by advancing a comprehensive theoretical model of vesicle dynamics. The model quantitatively captures the experimentally measured swell-burst parameters for single-component GUVs, and reveals that thermal fluctuations enable rate-dependent pore nucleation, driving the dynamics of the swell-burst cycles. We further extract constitutional scaling relationships between the pulsatile dynamics and GUV properties over multiple timescales. Our findings provide a fundamental framework that has the potential to guide future investigations on the nonequilibrium dynamics of vesicles under osmotic stress.


Assuntos
Pressão Osmótica , Estresse Fisiológico , Lipossomas Unilamelares/química , Dermoscopia , Difusão , Soluções Hipotônicas/química , Bicamadas Lipídicas/química , Modelos Biológicos , Fosfatidilcolinas/química , Sacarose/química , Termodinâmica
9.
Biochem Biophys Res Commun ; 482(3): 454-458, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28212731

RESUMO

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.


Assuntos
Antineoplásicos/farmacologia , Lactalbumina/farmacologia , Ácidos Oleicos/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Lactalbumina/química , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Ácidos Oleicos/química , Oncogenes , Inibidores de Proteassoma/farmacologia , Inibidores de Proteínas Quinases/farmacologia
10.
J Biol Chem ; 290(2): 918-25, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25451920

RESUMO

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.


Assuntos
Diferenciação Celular/genética , Proteínas da Matriz Extracelular/biossíntese , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrogênese , Colágeno Tipo II/biossíntese , Matriz Extracelular , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/biossíntese , Humanos , Fatores de Transcrição SOX9/biossíntese
11.
Langmuir ; 32(9): 2151-63, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26866787

RESUMO

Giant lipid vesicles are topologically closed compartments bounded by semipermeable flexible shells, which isolate femto- to picoliter quantities of the aqueous core from the surrounding bulk. Although water equilibrates readily across vesicular walls (10(-2)-10(-3) cm(3) cm(-2) s(-1)), the passive permeation of solutes is strongly hindered. Furthermore, because of their large volume compressibility (∼10(9)-10(10) N m(-2)) and area expansion (10(2)-10(3) mN m(-1)) moduli, coupled with low bending rigidities (10(-19) N m), vesicular shells bend readily but resist volume compression and tolerate only a limited area expansion (∼5%). Consequently, vesicles experiencing solute concentration gradients dissipate the available chemical energy through the osmotic movement of water, producing dramatic shape transformations driven by surface-area-volume changes and sustained by the incompressibility of water and the flexible membrane interface. Upon immersion in a hypertonic bath, an increased surface-area-volume ratio promotes large-scale morphological remodeling, reducing symmetry and stabilizing unusual shapes determined, at equilibrium, by the minimal bending-energy configurations. By contrast, when subjected to a hypotonic bath, walls of giant vesicles lose their thermal undulation, accumulate mechanical tension, and, beyond a threshold swelling, exhibit remarkable oscillatory swell-burst cycles, with the latter characterized by damped, periodic oscillations in vesicle size, membrane tension, and phase behavior. This cyclical pattern of the osmotic influx of water, pressure, membrane tension, pore formation, and solute efflux suggests quasi-homeostatic self-regulatory behavior allowing vesicular compartments produced from simple molecular components, namely, water, osmolytes, and lipids, to sense and regulate their microenvironment in a negative feedback loop.


Assuntos
Lipossomos/química , Modelos Químicos , Tensão Superficial
12.
Langmuir ; 32(21): 5445-50, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27164321

RESUMO

Using single-particle tracking, we investigate the interaction of small unilamellar vesicles (SUVs) that are electrostatically tethered to the freestanding membrane of a giant unilamellar vesicle (GUV). We find that the surface mobility of the GUV-riding SUVs is Brownian, insensitive to the bulk viscosity, vesicle size, and vesicle fluidity but strongly altered by the viscosity of the underlying membrane. Analyzing the diffusional behavior of SUVs within the Saffman-Delbrück model for the dynamics of membrane inclusions supports the notion that the mobility of the small vesicles is coupled to that of dynamically induced lipid clusters within the target GUV membrane. The reversible binding also offers a nonperturbative means for measuring the viscosity of biomembranes, which is an important parameter in cell physiology and function.

13.
J Biol Chem ; 288(24): 17460-71, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23629662

RESUMO

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.


Assuntos
Antineoplásicos/farmacologia , Lactalbumina/farmacologia , Ácido Oleico/farmacologia , Ácidos Oleicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células Jurkat , Lactalbumina/química , Metaboloma/efeitos dos fármacos , Ácido Oleico/química , Ácidos Oleicos/química , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma/efeitos dos fármacos
14.
Nat Chem ; 16(1): 54-62, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37414881

RESUMO

A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.


Assuntos
Separação de Fases , Lipossomas Unilamelares
15.
Membranes (Basel) ; 13(2)2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36837639

RESUMO

The stratum corneum (SC), the outer layer of the skin, plays a crucial role as a barrier protecting the underlying cells from external stress. The SC comprises three key components: ceramide (CER), free fatty acid (FFA), and cholesterol, along with small fractions of cholesterol sulfate and cholesterol ester. In order to gain a deeper understanding about the interdependence of the two major components, CER and FFA, on the organizational, structural, and functional properties of the SC layer, a library of SC lipid liposome (SCLL) models was developed by mixing CER (phytosphingosine or sphingosine), FFA (oleic acid, palmitic acid, or stearic acid), cholesterol, and cholesterol sulfate. Self-assembly of the SC lipids into lamellar phases was first confirmed by small-angle X-ray scattering. Short periodicity and long periodicity phases were identified for SCLLs containing phytosphingosines and sphingosine CERs, respectively. Furthermore, unsaturation in the CER acyl and FFA chains reduced the lipid conformational ordering and packing density of the liposomal bilayer, which were measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. The introduction of unsaturation in the CER and/or FFA chains also impacted the lamellar integrity and permeability. This extensive library of SCLL models exhibiting physiologically relevant lamellar phases with defined structural and functional properties may potentially be used as a model system for screening pharmaceuticals or cosmetic agents.

16.
Pharmaceutics ; 15(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36839864

RESUMO

Multidrug-resistant tuberculosis (MDR) continues to pose a threat to public health. Previously, we identified a cationic host defense peptide with activity against Mycobacterium tuberculosis in vivo and with a bactericidal effect against MDR M. tuberculosis at therapeutic concentrations. To understand the mechanisms of this peptide, we investigated its interactions with live M. tuberculosis and liposomes as a model. Peptide interactions with M. tuberculosis inner membranes induced tube-shaped membranous structures and massive vesicle formation, thus leading to bubbling cell death and ghost cell formation. Liposomal studies revealed that peptide insertion into inner membranes induced changes in the peptides' secondary structure and that the membranes were pulled such that they aggregated without permeabilization, suggesting that the peptide has a strong inner membrane affinity. Finally, the peptide targeted essential proteins in M. tuberculosis, such as 60 kDa chaperonins and elongation factor Tu, that are involved in mycolic acid synthesis and protein folding, which had an impact on bacterial proliferation. The observed multifaceted targeting provides additional support for the therapeutic potential of this peptide.

17.
ACS Bio Med Chem Au ; 2(1): 73-83, 2022 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-37102179

RESUMO

The role of an amphiphilic environment in the functional regulation of integral membrane proteins is well appreciated but how specific amphiphilic surrounding influences the conformational plasticity and function of a protein is less obvious. We focus on the Salmonella phosphoglycerate transport system (pgt)-encoded outer membrane protease E (PgtE), which plays an important role in tissue infiltration and survival of Salmonella enterica. Despite our understanding of its physiological functions, elucidation of its enzymatic behavior in response to the immediate amphiphilic surrounding is lacking. We monitor the proteolytic activity of PgtE reconstituted in Zwittergent 3-12 detergent micelles or a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer and examine factors that influence its activity. We find, to our surprise, that PgtE, which is thought to elicit a rapid response toward various substrates, showed hysteretic enzymatic behavior, characterized by a prominent lag phase prior to achieving the exponential steady state in its detergent-stabilized form as well as in the outer membrane embedded native state in live bacteria. The lag phase was abolished under three conditions: preformation of an inactive detergent-stabilized PgtE-substrate complex without lipopolysaccharide (LPS), LPS-bound detergent-stabilized PgtE that had reached steady state velocity, or PgtE reconstituted into a POPC bilayer environment. Interestingly, detergent- and bilayer-stabilized PgtE showed comparable steady-state activity. And strikingly, lipopolysaccharide (LPS) becomes nonessential for the activation of PgtE when the protein is reconstituted in the phospholipid bilayer, contrasting a long-standing notion that LPS is required for proteases belonging to the omptin family to be proteolytically active. These findings suggest intriguing biological nuances for the proteolytic function of PgtE that were not well appreciated previously and offer new perspectives that may generally be applicable for omptins.

18.
Biofactors ; 48(5): 1145-1159, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35388547

RESUMO

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.


Assuntos
Bicamadas Lipídicas , Neoplasias da Bexiga Urinária , Humanos , Lactalbumina/química , Lactalbumina/metabolismo , Lactalbumina/farmacologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Ácido Oleico/química , Ácido Oleico/metabolismo , Ácido Oleico/farmacologia , Fosfatidilcolinas/química , Esfingomielinas/química , Extratos de Tecidos , Lipossomas Unilamelares
19.
Mol Cell Oncol ; 8(5): 1974278, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34859140

RESUMO

The protein-lipid complex alpha1-oleate, derived from HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), is identified as a molecular entity with significant therapeutic potential. Structural characterization of the complex and results of a successful placebo-controlled clinical trial are presented.

20.
Nat Commun ; 12(1): 3427, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103518

RESUMO

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.


Assuntos
Peptídeos/química , Peptídeos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Determinação de Ponto Final , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Oleicos/química , Peptídeos/farmacologia , Placebos , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Termodinâmica , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
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