Double Hydrogen Bonding between Side Chain Carboxyl Groups in Aqueous Solutions of Poly (ß-L-Malic Acid): Implication for the Evolutionary Origin of Nucleic Acids.

The RNA world hypothesis holds that in the evolutionary events that led to the emergence of life RNA preceded proteins and DNA and is supported by the ability of RNA to act as both a genetic polymer and a catalyst. On the other hand, biosynthesis of nucleic acids requires a large number of enzymes and chemical synthesis of RNA under presumed prebiotic conditions is complicated and requires many sequential steps. These observations suggest that biosynthesis of RNA is the end product of a long evolutionary process. If so, what was the original polymer from which RNA and DNA evolved? In most syntheses of simpler RNA or DNA analogs, the D-ribose phosphate polymer backbone is altered and the purine and pyrimidine bases are retained for hydrogen bonding between complementary base pairs. However, the bases are themselves products of complex biosynthetic pathways and hence they too may have evolved from simpler polymer side chains that had the ability to form hydrogen bonds. We hypothesize that the earliest evolutionary predecessor of nucleic acids was the simple linear polyester, poly (ß-D-malic acid), for which the carboxyl side chains could form double hydrogen bonds. In this study, we show that in accord with this hypothesis a closely related polyester, poly (ß-L-malic acid), uses carboxyl side chains to form robust intramolecular double hydrogen bonds in moderately acidic solution.

The Hypothesis that the Genetic Code Originated in Coupled Synthesis of Proteins and the Evolutionary Predecessors of Nucleic Acids in Primitive Cells.

Although analysis of the genetic code has allowed explanations for its evolution to be proposed, little evidence exists in biochemistry and molecular biology to offer an explanation for the origin of the genetic code. In particular, two features of biology make the origin of the genetic code difficult to understand. First, nucleic acids are highly complicated polymers requiring numerous enzymes for biosynthesis. Secondly, proteins have a simple backbone with a set of 20 different amino acid side chains synthesized by a highly complicated ribosomal process in which mRNA sequences are read in triplets. Apparently, both nucleic acid and protein syntheses have extensive evolutionary histories. Supporting these processes is a complex metabolism and at the hub of metabolism are the carboxylic acid cycles. This paper advances the hypothesis that the earliest predecessor of the nucleic acids was a ß-linked polyester made from malic acid, a highly conserved metabolite in the carboxylic acid cycles. In the ß-linked polyester, the side chains are carboxylic acid groups capable of forming interstrand double hydrogen bonds. Evolution of the nucleic acids involved changes to the backbone and side chain of poly(ß-d-malic acid). Conversion of the side chain carboxylic acid into a carboxamide or a longer side chain bearing a carboxamide group, allowed information polymers to form amide pairs between polyester chains. Aminoacylation of the hydroxyl groups of malic acid and its derivatives with simple amino acids such as glycine and alanine allowed coupling of polyester synthesis and protein synthesis. Use of polypeptides containing glycine and l-alanine for activation of two different monomers with either glycine or l-alanine allowed simple coded autocatalytic synthesis of polyesters and polypeptides and established the first genetic code. A primitive cell capable of supporting electron transport, thioester synthesis, reduction reactions, and synthesis of polyesters and polypeptides is proposed. The cell consists of an iron-sulfide particle enclosed by tholin, a heterogeneous organic material that is produced by Miller-Urey type experiments that simulate conditions on the early Earth. As the synthesis of nucleic acids evolved from ß-linked polyesters, the singlet coding system for replication evolved into a four nucleotide/four amino acid process (AMP = aspartic acid, GMP = glycine, UMP = valine, CMP = alanine) and then into the triplet ribosomal process that permitted multiple copies of protein to be synthesized independent of replication. This hypothesis reconciles the "genetics first" and "metabolism first" approaches to the origin of life and explains why there are four bases in the genetic alphabet.

Evolution of the genetic code by incorporation of amino acids that improved or changed protein function.

Fifty years have passed since the genetic code was deciphered, but how the genetic code came into being has not been satisfactorily addressed. It is now widely accepted that the earliest genetic code did not encode all 20 amino acids found in the universal genetic code as some amino acids have complex biosynthetic pathways and likely were not available from the environment. Therefore, the genetic code evolved as pathways for synthesis of new amino acids became available. One hypothesis proposes that early in the evolution of the genetic code four amino acids-valine, alanine, aspartic acid, and glycine-were coded by GNC codons (N = any base) with the remaining codons being nonsense codons. The other sixteen amino acids were subsequently added to the genetic code by changing nonsense codons into sense codons for these amino acids. Improvement in protein function is presumed to be the driving force behind the evolution of the code, but how improved function was achieved by adding amino acids has not been examined. Based on an analysis of amino acid function in proteins, an evolutionary mechanism for expansion of the genetic code is described in which individual coded amino acids were replaced by new amino acids that used nonsense codons differing by one base change from the sense codons previously used. The improved or altered protein function afforded by the changes in amino acid function provided the selective advantage underlying the expansion of the genetic code. Analysis of amino acid properties and functions explains why amino acids are found in their respective positions in the genetic code.

Hsp90 and mitochondrial proteases Yme1 and Yta10/12 participate in ATP synthase assembly in Saccharomyces cerevisiae.

Hsc82 and Hsp82, the Hsp90 family proteins of yeast, are both required for fermentative growth at 37°C. Inactivation of either of the mitochondrial AAA proteases, Yme1 or Yta10/12, allows fermentative growth of hsc82∆ or hsp82∆ strains at 37°C. Genetic evidence indicates interaction of Hsc82/Hsp82 with the Yme1 and Yta10/Yta12 complexes in promoting F(1)F(o)-ATPase activity, with Hsc82 specifically required for F(1)-ATPase assembly. A previously reported mutation in Rpt3, one of the six ATPases of the proteasome, suppresses yme1∆ phenotypes and increases transcription of HSC82 but not HSP82. These genetic interactions describe a functional role for Hsp90 proteins in mitochondrial biogenesis.

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

The hinge region between two ubiquitin-like domains destabilizes recombinant ISG15 in solution.

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated within the endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15 resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution. It was hypothesized that the hinge region contributed to the instability of rbovISG15. Within 24 h of dialysis, rbovISG15 formed complexes as detected by reducing and denaturing SDS-PAGE. However, chemical perturbations of cysteine prevented formation of rbovISG15 complexes over time. Furthermore, a site-directed mutant of rbovISG15 (Cys80Ser) was isomeric and more stable than rbovISG15. Neither wild-type nor mutant rbovISG15 was able to interact with the ISG15 E1 initiating enzyme, UBE1L, in an in vitro pull-down assay. Ovine (ov) ISG15 has three additional amino acids within the hinge region that were hypothesized to increase stability and the degree of interaction with UBE1L because of increased separation of the ubiquitin-like domains. Over time in solution, rovISG15 the level of rovISG15 secondary structure was diminished, whereas the Cys80Ser rovISG15 structure did not change. A GST-Cys80Ser rovISG15 fusion protein had increased structural stability and enhanced protein-protein interaction with UBE1L after dialysis for 48 h, when compared to the GST-rovISG15 fusion protein or rbovISG15. Models of bovISG15, Cys80Ser bovISG15, and ovISG15 were constructed, which confirmed that the hinge region between the two ubiquitin domains destabilizes rbovISG15 in solution.

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme.

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta.

BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.

Biosynthesis and processing of Spodoptera frugiperda alpha-mannosidase III.

We previously cloned a lepidopteran insect cell cDNA that encodes a class II alpha-mannosidase that is localized in the Golgi apparatus but is cobalt-dependent, has a neutral pH optimum, hydrolyzes Man(5)GlcNAc(2) to Man(3)GlcNAc(2), and cannot hydrolyze GlcNAcMan(5)GlcNAc(2). This enzyme was designated SfManIII to distinguish it from Golgi alpha-mannosidase II and indicate its derivation from the fall armyworm Spodoptera frugiperda. In the present study, we prepared a polyclonal antibody and used it to study the biosynthesis and processing of SfManIII. The results showed that Sf9 cells produce at least three different forms of SfManIII. SfManIII is initially synthesized as a precursor glycoprotein, which is slowly converted to two smaller end products with at least some endoglycosidase H-resistant N-glycans. The smallest form of SfManIII is the only one of these two products that accumulates in the extracellular fraction. Tunicamycin blocked the production of SfManIII activity and the secretion of SfManIII protein and activity. Castanospermine blocked production of the larger SfManIII product, retarded production of the smaller, increased intracellular SfManIII activity, and decreased extracellular SfManIII activity. Together, these results indicate that SfManIII is initially synthesized as a high-mannose glycoprotein precursor, its N-glycans are trimmed as it is transported to the Golgi apparatus, and a subpopulation, which appears to be proteolytically cleaved, is secreted in enzymatically active form. N-glycosylation is required for the production of active SfManIII, and N-glycosylation and N-glycan trimming are both required for the efficient secretion of an active form of this protein.