Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics

Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts.     

Synaptic protein UNC-13 interacts with an F-box protein that may target it for degradation by proteasomes

UNC-13 protein participates in regulating neurotransmitter release. In Drosophila melanogaster, proteasomal degradation controls UNC-13 levels at synapses. Function of the amino-terminal region of a 207 kDa form of Caenorhabditis elegans UNC-13 is unknown. Yeast two-hybrid and secondary yeast assays identified an F-box protein that interacts with this amino-terminal region. As F-box proteins bind proteins targeted for proteasomal degradation, this protein may participate in degrading a subset of UNC-13 proteins, suggesting that different forms of UNC-13 are regulated differently. Yeast assays also identified an exonuclease, a predicted splicing factor, and a protein with coiled-coil domains, indicating that UNC-13 may affect RNA function.     

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.     

Semen displacement as a sperm competition strategy

Using a sample of 652 college students, we examined several implications of the hypothesis that the shape of the human penis evolved to enable males to substitute their semen for those of their rivals. The incidence of double mating by females appears sufficient to make semen displacement adaptive (e.g., one in four females acknowledge infidelity, one in eight admit having sex with two or more males in a 24-hour period, and one in 12 report involvement in one or more sexual threesomes with two males). We also document several changes in post-ejaculatory behavior (e.g., reduced thrusting, penis withdrawal, loss of an erection) which may have evolved to minimize displacement of the male’s own semen. Consistent with predictions derived from a theoretical model (Gallup and Burch 2006), we discovered that most females report waiting at least 48 hours following an instance of infidelity before resuming sex with their in-pair partners.     

Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts

Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.     

Comparative genomics of emerging human ehrlichiosis agents

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.     

Characterization of Angiotensin Converting Enzyme-2 (ACE2) in Human Urine

Angiotensin converting enzyme-2 (ACE2) is a recently described membrane-bound carboxypeptidase identified by its homology to ACE, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin II (Ang II). ACE2 inactivates Ang II and is thus thought to act in a counter-regulatory fashion to ACE. ACE2 is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. A soluble form of ACE, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ACE2 has been reported in cell culture, it is not yet known whether this occurs in vivo. The present study has identified ACE2 in human urine, both by a sensitive fluorescence-based activity assay and by Western immunoblot. Levels of ACE2 were surprisingly higher than ACE, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. Future studies will determine whether increased expression of ACE2 in renal diseases are reflected in higher urinary levels of this novel enzyme.Australian Peptide Conference Issue.