Strong and weak zinc binding sites in human zinc-α2-glycoprotein

Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the β-adrenergic receptor.     

Cranial morphological variation among contemporary Mexicans: Regional trends,ancestral affinities,and genetic comparisons

Genetic research has documented geographical variation within Mexico that corresponds to trends in ancestry admixture from postcolonial times on. The purpose of this study is to determine whether craniometric variation among contemporary Mexicans is comparable to that reported in genetic studies. Standard osteometric measurements were taken on 82 male crania derived from forensic cases, with geographic origins of the specimens spanning over two‐thirds of Mexico's states. To study similarities in regional clustering patterns with genetic data, k‐means clustering analyses were performed, followed by chi‐square tests of association between cluster assignments and geographic region of origin. Normal mixtures analyses were performed, centered on three “ancestral” sample proxies to estimate classification probability to each ancestry. The results demonstrate that the cranial morphological sample data cluster similarly to the regional groupings inferred from the genetic data. Additionally, the results indicate a gradient trend in population structure for contemporary Mexicans, with the proportion of Amerindian ancestry increasing from North to South while, conversely, European ancestry proportion estimates increase from South to North. Furthermore, the probabilities for classification of African ancestry remained low across the regions, again reflecting the results for the genetic data. Cranial morphological variation is well aligned with the genetic data for describing broad trends among Mexican populations, as well as yielding comparable estimates of general ancestry affiliations that reflect Mexico's history of Spanish contact and colonialism. Am J Phys Anthropol 151:506–517, 2013. © 2013 Wiley Periodicals, Inc.     

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.     

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.     

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K⁺ accumulation in the matrix.     

Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence

At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.     

Both spontaneous Ins2+/− and streptozotocin‐induced type I diabetes cause bone loss in young mice

The adolescent skeleton undergoes accelerated growth determining overall bone density, length, and quality. Diseases such as type 1 diabetes (T1D), most often diagnosed in adolescents, can alter bone processes and promote bone loss. Studies examining type 1 diabetic (T1D) bone pathologies typically utilize adult mice and rely on pharmacologic models such as streptozotocin (STZ)‐induced diabetic rodents. To test the effect of T1D on adolescent bone growth/density we used a novel juvenile genetic model (Ins2^+/? mice) that spontaneously develop T1D at approximately 5 weeks of age and compared our findings with STZ‐induced T1D mice. Compared to controls, both Ins2^+/? and STZ‐induced T1D mice displayed blood glucose levels greater than 300 mg/dl and reduced body, fat and muscle mass as well as femur trabecular bone density. STZ mice exhibited greater bone loss compared to Ins2^+/? mice despite having lower blood glucose levels. Cortical bone was affected in STZ but not Ins2^+/? mice. Osteocalcin serum protein and bone RNA levels decreased in both models. Consistent with studies in adult mice, STZ adolescent mice displayed increased marrow adiposity, however this was not observed in the Ins2^+/? mice. Reduced femur length, decreased growth plate thickness and decreased collagen II expression in both model simplies impaired cartilage formation. In summary, both pharmacologic and spontaneous adolescent T1D mice demonstrated a bone synthesis and growth defect. STZ appears to cause a more severe phenotype. Thus, the Ins2^+/? mouse could serve as a useful model to study adolescent T1D bone loss with fewer complications. J. Cell. Physiol. 228: 689–695, 2013. © 2012 Wiley Periodicals, Inc.     

Mutational Analysis of the Pseudomonas aeruginosa Myovirus ?KZ Morphogenetic Protease gp175

Pseudomonas aeruginosa myovirus ϕKZ has a 270-kb genome within a T=27 icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in ϕKZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.     

ICAM-2 and lipid rafts disappear from the basal plasma membrane of uterine epithelial cells during early pregnancy in rats

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.