The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

Siderophore production and outer membrane proteins of selected Vibrio vulnificus strains under conditions of iron limitation

Abstract The tonB gene product is necessary for the energy-dependent transport of ferric chelates and vitamin B₁₂ across the Escherichia coli outer membrane. When carried on multicopy plasmids, the cloned tonB gene complemented tonB hosts, restoring transport of ferri-siderophone complexes and vitamin B₁₂, and susceptibility to the group B colicins and phage ф80. The levels of these activities were all markedly lower than when the tonB ⁺ gene was present in single copy. This depression of TonB function occurred even when the chromosome carried the normal tonB ⁺ allele, but plasmids carrying only a portion of the tonB gene, including the 5'-regulatory region, were not inhibitory.     

The induction of valine dehydrogenase activity from Streptomyces by L-valine is not repressed by ammonium

Summary Activity of valine dehydrogenases (VDH) from Streptomyces aureofaciens and S. fradiae is strongly induced by L-valine even in the presence of 25mM NH₄ ⁺. When added into 16 h-old cultures growing with 100mM NH₄ ⁺, L-valine induced the synthesis of VDH. The results indicate that Streptomyces can utilize L-valine in the presence of NH₄ ⁺, and the induction of VDH activity by L-valine is not repressed by NH₄ ⁺.     

Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

The alpha1 subunit of GABAA receptor is repressed by c-myc and is pro-apoptotic

The c-myc oncoprotein plays a critical role in the regulation of cellular proliferation and apoptosis. To mediate these biological functions, a variety of target genes are activated or repressed by c-myc, but few genes have yet been identified that directly mediate c-myc's role in proliferation or apoptosis. During a screen for genes that are repressed by c-myc, we identified the alpha1 subunit of gamma aminobutyric acid receptor (GABAAR-alpha1) as a novel target of c-myc. GABAAR is the major inhibitory neurotransmitter receptor in the mammalian central nervous system and is involved in developmental events in the brain, such as neurite outgrowth, neuronal survival, neuronal migration, and proliferation. We show here that GABAAR-alpha1 expression is rapidly and directly repressed by c-myc. GABAAR-alpha1 expression is elevated in c-myc null cells and upregulation of GABAAR-alpha1 correlates with downregulation of c-myc protein expression during neuronal cell differentiation. We also show that overexpression of GABAAR-alpha1 causes apoptosis, which is blocked by the coexpression of Bcl-2 or Bcl-XL. Induction of apoptosis is specific for the alpha1 subunit, since neither the beta1 or beta2 subunits of GABAAR induced apoptosis. Derepression of GABAAR-alpha1 expression upon downregulation of c-myc represents a unique apoptotic mechanism and a distinct function for the alpha1 subunit, independent of its role as a component of the GABAAR in the plasma membrane. In addition, the regulation of GABAAR-alpha1 expression by c-myc provides a potential direct role for the Myc proteins in neurological processes and neurodegenerative disorders.     

Heme-dependent metalloregulation by the iron response regulator (Irr) protein in Rhizobium and other Alpha-proteobacteria

Perception and response to nutritional iron by bacteria is essential for viability, and for the ability to adapt to the environment. The iron response regulator (Irr) is part of a novel regulatory scheme employed by Rhizobium and other Alpha-Proteobacteria to control iron-dependent gene expression. Bradyrhizobium japonicum senses iron through the status of heme biosynthesis to regulate gene expression, thus it responds to an iron-dependent process rather than to iron directly. Irr mediates this response by interacting directly with ferrochelatase, the enzyme that catalyzes the final step in heme biosynthesis. Irr is expressed under iron limitation to both positively and negatively modulate gene expression, but degrades in response to direct binding to heme in iron-sufficient cells. Studies with Rhizobium reveal that the regulation of iron homeostasis in bacteria is more diverse than has been generally assumed.     

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase

The new discipline of Evolutionary Developmental Biology (Evo-Devo) is facing the fascinating paradox of explaining morphological evolution using conserved pieces or genes to build divergent animals. The cephalochordate amphioxus is the closest living relative to the vertebrates, with a simple, chordate body plan, and a genome directly descended from the ancestor prior to the genome-wide duplications that occurred close to the origin of vertebrates. Amphioxus morphology may have remained relatively invariant since the divergence from the vertebrate lineage, but the amphioxus genome has not escaped evolution. We report the isolation of a second Emx gene (AmphiEmxB) arising from an independent duplication in the amphioxus genome. We also argue that a tandem duplication probably occurred in the Posterior part of the Hox cluster in amphioxus, giving rise to AmphiHox14, and discuss the structure of the chordate and vertebrate ancestral clusters. Also, a tandem duplication of Evx in the amphioxus lineage produced a prototypical Evx gene (AmphiEvxA) and a divergent gene (AmphiEvxB), no longer involved in typical Evx functions. These examples of specific gene duplications in amphioxus, and other previously reported duplications summarized here, emphasize the fact that amphioxus is not the ancestor of the vertebrates but 'only' the closest living relative to the ancestor, with a mix of prototypical and amphioxus-specific features in its genome.     

The axon degeneration gene SARM1 is evolutionarily distinct from other TIR domain-containing proteins

Many forms of neurodegenerative disease are characterized by Wallerian degeneration, an active program of axonal destruction. Recently, the important player which enacts Wallerian degeneration was discovered, the multidomain protein SARM1. Since the SARM1 protein has classically been thought of as an innate immune molecule, its role in Wallerian degeneration has raised questions on the evolutionary forces acting on it. Here, we synthesize a picture of SARM1’s evolution through various organisms by examining the molecular and genetic changes of SARM1 and the genes around it. Using proteins that possess domains homologous to SARM1, we established distances and Ka/Ks values through 5671 pairwise species–species comparisons. We demonstrate that SARM1 diverged across species in a pattern similar to other SAM domain-containing proteins. This is surprising, because it was expected that SARM1 would behave more like its TIR domain relatives. Going along with this divorce from TIR, we also noted that SARM1’s TIR is under stronger purifying selection than the rest of the TIR domain-containing proteins (remaining highly conserved). In addition, SARM1’s synteny analysis reveals that the surrounding gene cluster is highly conserved, functioning as a potential nexus of gene functionality across species. Taken together, SARM1 demonstrates a unique evolutionary pattern, separate from the TIR domain protein family.     

E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products

Induction of apoptosis seems to be a key function in maintaining normal cell growth by exerting negative controls on cell proliferation and suppressing tumorigenesis. The adenovirus E1A oncogene shows both cell cycle progression and apoptotic functions. To understand the mechanism of E1A-induced apoptosis, the apoptotic function of E1A 13S was investigated in p53-null cells. We show here that E1A is sufficient by itself to induce substantial apoptosis independent of p53 and other adenoviral genes. The apoptotic function of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose)-polymerase. Cell death is significantly blocked by the caspase inhibitor zVAD-fmk and when coexpressed with E1B19K, Bcl-2 or the retinoblastoma protein (RB). Analyses of E1A mutants indicated that the apoptotic activity of E1A correlates closely with the ability to bind the key regulators of E2F1-induced apoptosis, p300 and RB. Finally, in vivo relevance of down-modulation of p53-independent apoptosis for efficient transformation is demonstrated.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation

Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)-protein complex, CP43', which is encoded by the isiA gene. The deduced amino-acid sequence of CP43' predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43' remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA cells). Compared with isiA cells, under iron deprivation, wild-type cells showed both lower rates of photosystem II-mediated O2 evolution at limiting light irradiances and decreased yields of room temperature Chl fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild-type cells with CP43' is attributable to increased non-radiative dissipation of light energy. In agreement with this hypothesis, isiA cells were more susceptible to photoinhibition of photosynthesis than wild-type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43' functions as a non-radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.