Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration.

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.

Prebiotic Protocell Membranes Retain Encapsulated Contents during Flocculation, and Phospholipids Preserve Encapsulation during Dehydration.

The first cell membranes were likely composed of single-chain amphiphiles such as fatty acids. An open question is whether fatty acid membranes could have functioned within evaporative lakes on the early Earth, which have been hypothesized to concentrate prebiotic reactants. Evaporation also concentrates monovalent salts, which in turn cause fatty acid membrane vesicles to flocculate; significant loss of encapsulated contents during flocculation would have impeded early cell evolution. Here, we tested whether fatty acid vesicles retain encapsulated contents after flocculation and after drying. We found that vesicles composed of 2:1 decanoic acid:decanol encapsulate calcein dye throughout a process of flocculation in saturated salt solution and subsequent disaggregation of vesicles by dilution of the salt. However, 30 minutes of complete dehydration disrupted encapsulation by fatty acid vesicles. In contrast, phospholipid vesicles maintained encapsulation. Our results reveal a selective pressure for protocells to incorporate phospholipids: while fatty acid membranes can retain encapsulated contents during periods of dilute and saturating salt, phospholipids are necessary for encapsulation during dry periods. Our results are consistent with the hypothesis that evaporative lakes were productive sites for prebiotic chemistry and the origin of cells.

Prebiotic Vesicles Retain Solutes and Grow by Micelle Addition after Brief Cooling below the Membrane Melting Temperature.

Replication of RNA genomes within membrane vesicles may have been a critical step in the development of protocells on the early Earth. Cold temperatures near 0 °C improve the stability of RNA and allow efficient copying, while some climate models suggest a cold early Earth, so the first protocells may have arisen in cold-temperature environments. However, at cold temperatures, saturated fatty acids, which would have been available on the early Earth, form gel-phase membranes that are rigid and restrict mobility within the bilayer. Two primary roles of protocell membranes are to encapsulate solutes and to grow by incorporating additional fatty acids from the environment. We test here whether fatty acid membranes in the gel phase accomplish these roles. We find that gel-phase membranes of 10-carbon amphiphiles near 0 °C encapsulate aqueous dye molecules as efficiently as fluid-phase membranes do, but the contents are released if the aqueous solution is frozen at -20 °C. Gel-phase membranes do not grow measurably by micelle addition, but growth resumes when membranes are warmed above the gel-liquid transition temperature. We find that longer, 12-carbon amphiphiles do not retain encapsulated contents near 0 °C. Together, our results suggest that protocells could have developed within environments that experience temporary cooling below the membrane melting temperature, and that membranes composed of relatively short-chain fatty acids would encapsulate solutes more efficiently as temperatures approached 0 °C.

Growth of Prebiotically Plausible Fatty Acid Vesicles Proceeds in the Presence of Prebiotic Amino Acids, Dipeptides, Sugars, and Nucleic Acid Components.

Fatty acid vesicles may have played a role in the origin of life as a major structural component of protocells, with the potential for encapsulation of genetic materials. Vesicles that grew and divided more rapidly than other vesicles could have had a selective advantage. Fatty acid vesicles grow by incorporating additional fatty acids from micelles, and certain prebiotic molecules (e.g., sugars, nucleobases, and amino acids) can bind to fatty acid vesicles and stabilize them. Here, we investigated whether the presence of a variety of biomolecules affects the overall growth of vesicles composed of decanoic acid, a prebiotically plausible fatty acid, upon micelle addition. We tested 31 molecules, including 15 dipeptides, 7 amino acids, 6 nucleobases or nucleosides, and 3 sugars. We find that the initial radius and final radius of vesicles are largely unaffected by the presence of the additional compounds. However, three dipeptides enhanced the initial rates of growth compared to control vesicles with no small molecules added; another three dipeptides decreased the initial rates of growth. We conclude that vesicles can indeed grow in the presence of a wide range of molecules likely to have been involved in the origin of life. These results imply that vesicles would have been able to grow in complex and heterogeneous chemical environments. We find that the molecules that enhance the initial growth rate tend to have hydrophobic groups (e.g., leucine), which may interact with the lipid membrane to affect growth rate; furthermore, the molecules that cause the largest decrease in initial growth rate are dipeptides containing a serine residue, which contains a hydroxyl group that could potentially hydrogen-bond with the fatty acid carboxylate groups.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells.

Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells.

Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury.

OBJECTIVE: Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage. METHODS: Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin-neutralizing monoclonal antibody (Scl-Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl-Ab or intraarticular (IA) delivery of a sclerostin antibody-Fab (Scl-Fab) fragment. RESULTS: Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin-KO mice showed no difference in histopathologic features of the knee joint compared to age-matched wild-type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl-Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl-Ab or IA injection of Scl-Fab had no effect on lesion development or severity. CONCLUSION: Genetic absence of sclerostin does not alter the normal development of age-dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl-Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.

Natural soda lakes provide compatible conditions for RNA and membrane function that could have enabled the origin of life.

The origin of life likely occurred within environments that concentrated cellular precursors and enabled their co-assembly into cells. Soda lakes (those dominated by Na+ ions and carbonate species) can concentrate precursors of RNA and membranes, such as phosphate, cyanide, and fatty acids. Subsequent assembly of RNA and membranes into cells is a long-standing problem because RNA function requires divalent cations, e.g. Mg2+, but Mg2+ disrupts fatty acid membranes. The low solubility of Mg-containing carbonates limits soda lakes to moderate Mg2+ concentrations (∼1 mM), so we investigated whether both RNAs and membranes function within these lakes. We collected water from Last Chance Lake and Goodenough Lake in Canada. Because we sampled after seasonal evaporation, the lake water contained ∼1 M Na+ and ∼1 mM Mg2+ near pH 10. In the laboratory, nonenzymatic, RNA-templated polymerization of 2-aminoimidazole-activated ribonucleotides occurred at comparable rates in lake water and standard laboratory conditions (50 mM MgCl2, pH 8). Additionally, we found that a ligase ribozyme that uses oligonucleotide substrates activated with 2-aminoimidazole was active in lake water after adjusting pH from ∼10 to 9. We also observed that decanoic acid and decanol assembled into vesicles in a dilute solution that resembled lake water after seasonal rains, and that those vesicles retained encapsulated solutes despite salt-induced flocculation when the external solution was replaced with dry-season lake water. By identifying compatible conditions for nonenzymatic and ribozyme-catalyzed RNA assembly, and for encapsulation by membranes, our results suggest that soda lakes could have enabled cellular life to emerge on Earth, and perhaps elsewhere.

TIMP3 checks inflammation.

Mice deficient in the metalloprotease inhibitor TIMP3, which inhibits the tumor-necrosis factor alpha (TNF-alpha)-converting enzyme (TACE, also called ADAM17), have elevated levels of TNF and severe inflammation in the liver. This result confirms the physiological importance of the soluble form of TNF and identifies TIMP3 as a crucial regulator of this inflammatory cytokine.

Plausible Sources of Membrane-Forming Fatty Acids on the Early Earth: A Review of the Literature and an Estimation of Amounts.

The first cells were plausibly bounded by membranes assembled from fatty acids with at least 8 carbons. Although the presence of fatty acids on the early Earth is widely assumed within the astrobiology community, there is no consensus regarding their origin and abundance. In this Review, we highlight three possible sources of fatty acids: (1) delivery by carbonaceous meteorites, (2) synthesis on metals delivered by impactors, and (3) electrochemical synthesis by spark discharges. We also discuss fatty acid synthesis by UV or particle irradiation, gas-phase ion-molecule reactions, and aqueous redox reactions. We compare estimates for the total mass of fatty acids supplied to Earth by each source during the Hadean eon after an extremely massive asteroid impact that would have reset Earth's fatty acid inventory. We find that synthesis on iron-rich surfaces derived from the massive impactor in contact with an impact-generated reducing atmosphere could have contributed ∼102 times more total mass of fatty acids than subsequent delivery by either carbonaceous meteorites or electrochemical synthesis. Additionally, we estimate that a single carbonaceous meteorite would not deliver a high enough concentration of fatty acids (∼15 mM for decanoic acid) into an existing body of water on the Earth's surface to spontaneously form membranes unless the fatty acids were further concentrated by another mechanism, such as subsequent evaporation of the water. Our estimates rely heavily on various assumptions, leading to significant uncertainties; nevertheless, these estimates provide rough order-of-magnitude comparisons of various sources of fatty acids on the early Earth. We also suggest specific experiments to improve future estimates. Our calculations support the view that fatty acids would have been available on the early Earth. Further investigation is needed to assess the mechanisms by which fatty acids could have been concentrated sufficiently to assemble into membranes during the origin of life.

Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels.

Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.

γ-Secretase-mediated growth hormone receptor proteolysis: mapping of the intramembranous cleavage site.

GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (α-secretase) and γ-secretase activities. α-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of γ-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential α- and γ-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that γ-secretase cleavage occurs at an ε-site in GHR's transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both α- and γ-secretase-mediated proteolysis and deletion of four residues around the ε-site precluded surface GHR expression and proteolysis. However, point mutations in and around the ε-site affected neither α- or γ-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (α-secretase followed by γ-secretase) and that ε-site cleavage by γ-secretase does not require a consensus sequence.

Binding of Dipeptides to Fatty Acid Membranes Explains Their Colocalization in Protocells but Does Not Select for Them Relative to Unjoined Amino Acids.

Dipeptides, which consist of two amino acids joined by a peptide bond, have been shown to have catalytic functions. This observation leads to fundamental questions relevant to the origin of life. How could peptides have become colocalized with the first protocells? Which structural features would have determined the association of amino acids and peptides with membranes? Could the association of dipeptides with protocell membranes have driven molecular evolution, favoring dipeptides over individual amino acids? Using pulsed-field gradient nuclear magnetic resonance, we find that several prebiotic amino acids and dipeptides bind to prebiotic membranes. For amino acids, the side chains and carboxylate contribute to the interaction. For dipeptides, the extent of binding is generally less than that of the constituent amino acids, implying that other mechanisms would be necessary to drive molecular evolution. Nevertheless, our results are consistent with a scheme in which the building blocks of the biological polymers colocalized with protocells prior to the emergence of RNA and proteins.

Tissue inhibitor of metalloproteinase-1 moderates airway re-epithelialization by regulating matrilysin activity.

Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.

Novel protein therapeutic joint retention strategy based on collagen-binding Avimers.

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.

Janus kinase 2 influences growth hormone receptor metalloproteolysis.

GH signals through the GH receptor (GHR), a cytokine receptor superfamily member that couples to the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2). In addition to its role in signaling, we recently implicated JAK2 in the regulation of cell surface GHR abundance by modulation of GHR trafficking and mature GHR stability. GHR is a target for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. We previously found that metalloprotease cleavage of GHR is dramatically lessened in fibroblasts derived from mice with targeted deletion of the zinc-binding domain of TNF-alpha-cleaving enzyme [TACE; ADAM17 (a disintegrin and metalloprotease)], implicating this transmembrane ectoenzyme as a GHR metalloprotease. In this study we used a human fibrosarcoma reconstitution system to compare the effects of RNA interference-mediated knockdown of TACE vs. a related metalloprotease, ADAM10. We found that TACE knockdown dramatically reduced both the pace and the degree of inducible GHR proteolysis and augmented the abundance of mature GHR, suggesting a role for TACE in constitutive receptor proteolysis in this system as well. Notably, ADAM10 knockdown also reduced inducible GHR proteolysis, although to a lesser degree than TACE knockdown, suggesting a contribution from this metalloprotease also. To determine whether JAK2 affects GHR proteolysis, we compared JAK2-deficient vs. JAK2-replete cells and found that phorbol 12-methyl 13-acetate-induced GHR proteolysis was significantly diminished in cells that lacked JAK2. Reconstitution with a GHR mutant that lacks the box 1 region (which mediates JAK2 association) resulted in phorbol 12-methyl 13-acetate-induced proteolysis similar in degree to that of the wild-type GHR in JAK2-deficient cells. Introduction of JAK2 did not affect the proteolysis of this box 1-deleted GHR, suggesting GHR-JAK2 association is required for JAK2 to affect GHR proteolysis. Additionally, the inhibitory effect of anti-GHRext-mAb, a conformation-sensitive GHR antibody, on receptor proteolysis was lost in cells that lacked JAK2. Our data indicate that the susceptibility of GHR to proteolysis is substantially affected by JAK2, suggesting yet another role for this kinase in determining GH sensitivity.

A Self-Assembled Aggregate Composed of a Fatty Acid Membrane and the Building Blocks of Biological Polymers Provides a First Step in the Emergence of Protocells.

We propose that the first step in the origin of cellular life on Earth was the self-assembly of fatty acids with the building blocks of RNA and protein, resulting in a stable aggregate. This scheme provides explanations for the selection and concentration of the prebiotic components of cells; the stabilization and growth of early membranes; the catalysis of biopolymer synthesis; and the co-localization of membranes, RNA and protein. In this article, we review the evidence and rationale for the formation of the proposed aggregate: (i) the well-established phenomenon of self-assembly of fatty acids to form vesicles; (ii) our published evidence that nucleobases and sugars bind to and stabilize such vesicles; and (iii) the reasons why amino acids likely do so as well. We then explain how the conformational constraints and altered chemical environment due to binding of the components to the membrane could facilitate the formation of nucleosides, oligonucleotides and peptides. We conclude by discussing how the resulting oligomers, even if short and random, could have increased vesicle stability and growth more than their building blocks did, and how competition among these vesicles could have led to longer polymers with complex functions.

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site.

GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.