Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.     

LB1 and LB6 Homo floresiensis are not modern human (Homo sapiens) cretins

Excavations in the late Pleistocene deposits at Liang Bua cave, Flores, have uncovered the skeletal remains of several small-bodied and small-brained hominins in association with stone artefacts and the bones of Stegodon. Due to their combination of plesiomorphic, unique and derived traits, they were ascribed to a new species, Homo floresiensis, which, along with Stegodon, appears to have become extinct ∼17 ka (thousand years ago). However, recently it has been argued that several characteristics of H. floresiensis were consistent with dwarfism and evidence of delayed development in modern human (Homo sapiens) myxoedematous endemic (ME) cretins. This research compares the skeletal and dental morphology in H. floresiensis with the clinical and osteological indicators of cretinism, and the traits that have been argued to be associated with ME cretinism in LB1 and LB6. Contrary to published claims, morphological and statistical comparisons did not identify the distinctive skeletal and dental indicators of cretinism in LB1 or LB6 H. floresiensis. Brain mass, skeletal proportions, epiphyseal union, orofacial morphology, dental development, size of the pituitary fossa and development of the paranasal sinuses, vault bone thickness and dimensions of the hands and feet all distinguish H. floresiensis from modern humans with ME cretinism. The research team responsible for the diagnosis of ME cretinism had not examined the original H. floresiensis skeletal materials, and perhaps, as a result, their research confused taphonomic damage with evidence of disease, and thus contained critical errors of fact and interpretation. Behavioural scenarios attempting to explain the presence of cretinous H. sapiens in the Liang Bua Pleistocene deposits, but not unaffected H. sapiens, are both unnecessary and not supported by the available archaeological and geochronological evidence from Flores.     

Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity

A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.     

Impact of the C-terminal loop of histidine triad nucleotide binding protein1 (Hint1) on substrate specificity

Although highly sequence similar, human histidine triad nucleotide binding protein (hHint1) and E. coli hinT (echinT) exhibit significant differences in their phosphoramidase substrate specificity and lysyl-adenylate hydrolytic activity. Observing that the C termini of each enzyme are highly dissimilar, we created two chimeric Hint's: one in which the C terminus of hHint1 was replaced with the C terminus of echinT (Hs/ec) and the other in which the C terminus of echinT was replaced with the C terminus of hHint1 (ec/Hs). The Hs/ec chimera exhibited nearly identical specificity constants (kcat/Km) to those found for echinT, whereas the specificity constants of the ec/Hs chimera were found to approximate those for hHint1. In particular, as observed for echinT, the Hs/ec chimera does not exhibit a preference for phosphoramidates containing d- or l- tryptophan, while the ec/Hs chimera adopts the human enzyme preference for the l configuration. In addition, the studies with each chimera revealed that differences in the ability of hHint1 and echinT to hydrolyze lysyl-AMP generated by either E. coli or human lysyl-tRNA synthetase were partially transferable by C-terminal loop exchange. Hence, our results support the critical role of the C-terminal loop of human and E. coli Hint1 on governing substrate specificity.     

Mutagenesis of apyrase conserved region 1 alters the nucleotide substrate specificity

Two apyrases having different substrate specificity, MP67 and MpAPY2, are present in Mimosa pudica. The substrate specificity of MP67 is quite high against ADP, and is distinct from any other apyrase. This might be attributed to the nucleotide binding motif (DXG) in apyrase conserved region 1. We performed a single amino acid substitution at position X in the motif. The ratio of the velocity of ATP/ADP hydrolysis was higher (approximately 1) for the S63A-MP67 mutant than for wild type-MP67 (0.19). Binding affinity for ADP of A75S-MpAPY2 mutant was increased to a level higher than that of the wild type MpAPY2. Thus, the residue at position X in the DXG motif plays an important role in determining nucleotide preference.     

Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Escherichia coli K12 is a bifunctional enzyme that catalyzes both the acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthetic pathway. In this study, we report the broad substrate specificity of the pyrophosphorylase domain of GlmU during its uridyltransfer reaction and the substrate priority is ranked in the following order: UTP > dUTP > dTTP >> CTP > dATP/dm⁶ ATP. This pyrophosphorylase domain of GlmU is also a tool to synthesize UDP-GlcNAc analogs, two examples of which were synthesized herein in multiple mg scale in vitro.     

Sutural complexity in artificially deformed human (Homo sapiens) crania.

The pattern of complexity of cranial sutures is highly variable both among and within species. Intentional cranial vault deformation in human populations provides a controlled natural experiment by which we were able to quantify aspects of sutural complexity and examine the relationship between sutural patterns and mechanical loading. Measures of sutural complexity (interdigitation, number, and size of sutural bones) were quantified from digitized tracings of 13 sutures and compared among three groups of crania (n = 70) from pre-European contact Peru. These groups represent sample populations deformed in 1) anteroposterior (AP) and 2) circumferential (C) directions and 3) an undeformed population. Intergroup comparisons show few differences in degree or asymmetry of sutural interdigitation. In the few comparisons which show differences, the C group is always more interdigitated than the other two while the AP group has more sutural bones. The sutures surrounding the temporal bone (sphenotemporal, occipitotemporal, and temporoparietal) most frequently show significant differences among groups. These differences are related to the more extreme binding of C type deformation and are consistent with hypothesized increases in tension at coronally oriented sutures in this group. The larger number of sutural bones in the AP group is consistent with the general broadening of the cranium in this group and with experimental evidence indicating the development of ossicles in areas of tension. We suggest that so few changes in sutural complexity occurred either because the magnitude of the growth vectors, unlike their direction, is not substantially altered or because mechanisms other than sutural growth modification are responsible for producing the altered vault shapes. In addition, the presence of fontanelles in the infant skulls during binding and the static nature of the binding may have contributed to the similarity in complexity among groups.     

Hypertrophy of the acetabulo-cristal buttress in Homo sapiens

In the early 1970s, excavation at the King site, a contact period Mississippian village in northwest Georgia, yielded the skeletal remains of a robust male (King 65) possessing marked hypertrophy of the acetabulo-cristal buttress. The buttress is morphologically similar to that of Plio-Pleistocene Homo but it is accompanied by an anatomically modern degree of thickening of the gluteal table of the ilium. Although the degree of cortical thickness of the gluteal table of the ilium is apparently species-specific, hypertrophy of the acetabulo-cristal buttress is developmental and may be expressed in all species of Homo. © 1993 Wiley-Liss, Inc.     

Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens

The protein Bc059385, whose solution structure is reported here, is the human representative of a recently identified family of membrane-anchored ubiquitin-fold (MUB) proteins. Analysis of their similarity to ubiquitin indicates that homologous amino acid residues in MUBs form a hydrophobic surface very similar to the recognition patch surrounding Ile-44 in ubiquitin. This suggests that MUBs may interact with proteins containing an alpha-helical motif similar to those of some ubiquitin binding domains. A disordered loop common to MUBs may also provide a second protein interaction site. From the available data, it is probable that this protein is prenylated and associated with the membrane. With <20% identity to ubiquitin, the MUB family further expands the sequence space that maps to the beta-grasp fold, and adds membrane localization to its list of functional roles.     

The complete complement of C1q-domain-containing proteins in Homo sapiens

The C-terminal domains of the A, B, C chains of C1q subcomponent of C1 complex represent a common structural motif, the C1q domain, that is found in a diverse range of proteins. We analyzed the human genome for the complete complement of this family and have identified a total of 31 independent gene sequences. The predominant organization of C1q-domain-containing (C1qDC) proteins includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 15 highly conserved residues within the C1q domain, among which 8 are invariant within the human gene set and these are predicted to cluster within the hydrophobic core of the protein. We suggest a 3-subfamily classification based on sequence homology. For some C1qDC-encoding genes, strict orthology has been retained throughout vertebrate evolution and these examples suggest a highly specific functional role for C1qDC proteins that has been under significant selective pressure. Alternatively, individual species have co-opted C1qDC proteins for roles that are highly specific to their biology, suggesting an evolutionary strategy of gene duplication and functional diversification. A more extensive analysis of the evolutionary relationship of C1qDC proteins reveals an ancient rooting, with clear members found in eubacterial species. Curiously, we have been unable to identify C1qDC-encoding genes in many eukaryotic genomcs, such as Sacchromyces cerivisae and C. elegans, suggesting that the retention or loss of this gene family throughout evolution has been sporadic.     

Rethinking the dispersal of Homo sapiens out of Africa

Current fossil, genetic, and archeological data indicate that Homo sapiens originated in Africa in the late Middle Pleistocene. By the end of the Late Pleistocene, our species was distributed across every continent except Antarctica, setting the foundations for the subsequent demographic and cultural changes of the Holocene. The intervening processes remain intensely debated and a key theme in hominin evolutionary studies. We review archeological, fossil, environmental, and genetic data to evaluate the current state of knowledge on the dispersal of Homo sapiens out of Africa. The emerging picture of the dispersal process suggests dynamic behavioral variability, complex interactions between populations, and an intricate genetic and cultural legacy. This evolutionary and historical complexity challenges simple narratives and suggests that hybrid models and the testing of explicit hypotheses are required to understand the expansion of Homo sapiens into Eurasia.     

Genetic modulation of the senescent phenotype in Homo sapiens

While it is important to search for unifying mechanisms of aging among a variety of model systems, evolutionary arguments suggest that the pathophysiological details of senescence may be, to some extent, species specific. Moreover, in species that are characterized by extensive genetic heterogeneity, such as our own, one is likely to find kindreds with both "private" and "public" markers of aging. Crude estimates of the number of loci with the potential to modulate aspects of the senescent phenotype of man suggest that thousands of genes could be involved. No single locus appears to modulate all features. Some affect predominantly a single aspect ("unimodal progeroid syndromes"); familial Alzheimer's disease is discussed as a prototype. Linkage studies indicate genetic heterogeneity for autosomal dominant forms of the disease. Some loci affect multiple aspects of the phenotype ("segmental progeroid disorders"); the prototype is Werner's syndrome, an autosomal recessive. Cells from homozygotes behave like mutator strains and undergo accelerated senescence in vitro. Elucidation of the biochemical genetic basis of such abiotrophic disorders may shed light on specific aging processes in man.     

现代人起源问题的新争论

现代人在分类学上称为晚期智人。他们是怎样起源的?长期来有两种理论,一种叫直接演化说(Direct evolution hypothesis)或系统说,也叫多地区起源说。这种理论认为现代人是由当地的早期智人以至猿人演化而来的,各人种在很久以前即已分歧,各自平行发展,演化成现代人,但长时期来互相有基因的交流。例如欧洲的白种人是由当地的尼安德特人逐渐演变来的;亚洲的黄种人是由当地的早期智人和猿人演变来的。另一种理论叫入侵论(Invasion hypothesis),也叫迁徙说或代替说,这种理论认为欧洲是典型尼人的家乡, 而解剖结构上的现代人是在欧洲以外地区演化出来, 然后侵人欧洲的尼人区域, 消灭了土著的尼人而形成的;侵人亚洲的也一样, 形成现代的黄种人。这种单一地区起源说过去一般认为亚洲西部是现代人最早形成的地区, 近年来有人提出非洲南部才是现代人最早形成的地区, 然后迁徙到世界各地。     

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.     

Recognition of facial expressions in a Japanese monkey (Macaca fuscata) and humans (Homo sapiens)

Recognition of facial expressions by a Japanese monkey and two humans was studied. The monkey subject matched 20 photographs of monkey facial expressions and 20 photographs of human facial expressions. Humans sorted the same pictures. Matching accuracy by the monkey was about 80% correct for both human and monkey facial expressions. The confusion matrices of those facial expressions were analyzed by a multi-dimensional scaling procedure (MDSCAL). The resulting MDS plots suggested that the important cues in recognizing facial expressions of monkeys were “thrusting the mouth” and ‘raising the eyebrows.” Comparison of the MDS plots by the monkey subject with those by human subjects suggested that the monkey categorized the human “happiness” faces. This may suggest that the monkey has an ability to recognize human smile face even though it is learned. However, the monkey did not differentiate the human “anger/disgust” faces from the human “sad” faces, while human subjects clearly did. This may correlate with the lack of eyebrow movement in monkeys.     

Structural similarity-based predictions of protein interactions between HIV-1 and Homo sapiens

Background

Vaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt3 ligand (FL) by recombinant vaccinia virus (rVACV) highly influenced properties of the virus in dependence on the level of expression.

Results

High production of FL driven by the strong synthetic promoter decreased the growth of rVACV in macrophage cell line J774.G8 in vitro as well as its multiplication in vivo when inoculated in mice. The inhibition of replication in vivo was mirrored in low levels of antibodies against vaccinia virus (anti-VACV) which nearly approached to the negative serum level in non-infected mice. Strong FL expression changed not only the host range of the recombinant but also the basic protein contents of virions. The major proteins - H3L and D8L - which are responsible for the virus binding to the cells, and 28 K protein that serves as a virulence factor, were changed in the membrane portion of P13-E/L-FL viral particles. The core virion fraction contained multiple larger, uncleaved proteins and a higher amount of cellular proteins compared to the control virus. The overexpression of FL also resulted in its incorporation into the viral core of P13-E/L-FL IMV particles. In contrary to the equimolar ratio of glycosylated and nonglycosylated FL forms found in cells transfected with the expression plasmid, the recombinant virus incorporated mainly the smaller, nonglycosylated FL.

Conclusions

It has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.