The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K 2 TeO 3 at 128 g/ml, compared to the MIC of 2 g/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a ϳ20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.Tellurite (TeO 3 2Ϫ ) resistance, which is found in many microorganisms (37), is carried on plasmids of IncHI, IncHII, and IncP incompatibility groups (3, 36, 39) and/or on chromosomes (9, 10, 29, 33). Tellurite resistance (Te r ) determinants are very diverse, and at least five different determinants have been identified (37). Te r determinants are unrelated, and probably different resistance mechanisms are involved. Except for that encoded by the Pseudomonas syringae tpm gene, Te r mechanisms are not clearly understood. tpm encodes a methyltransferase that catalyzes S-adenosyl-L-methionine (SAM) methylation of 6-mercaptopurine, a substrate for human thiopurine methyltransferase. It was assumed that tellurite is also a substrate for this enzyme (11).The Te r determinant tehAB, an operon located at 32.3 min on the Escherichia coli K-12 chromosome (38), encodes proteins of 330 (TehA) and 197 (TehB) amino acids. TehA is a membrane protein with 10 transmembrane segments, whereas TehB is a soluble protein. The closest homologues of these two genes are found in Haemophilus influenzae (15).A sequence search performed with the BLAST program (National Center for Biotechnology Information), followed by alignment analysis with the Genetics Computer Group software package (University of Wisconsin) (12), demonstrated that TehB displays some amino acid sequence similarity to many SAM-dependent non-nucleic acid methyltransferases. These proteins have three shared motifs, and the TehB proteins show homologies within all regions with comparable sequence interval distances (Fig.

Defects in the 20S/26S proteasome and conformational changes in alpha-synuclein (alpha-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether alpha-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects alpha-syn solubility and localization. Although alpha-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce alpha-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, alpha-syn. Concomitantly this treatment generated cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of alpha-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.

Proteasomal dysfunction has been suggested to contribute to the degeneration of nigrostriatal dopamine neurons in Parkinson's disease. A recent study reported that systemic treatment of rats with the proteasome inhibitor Z-lle-Glu(OtBu)-Ala-Leu-al (PSI) causes a slowly progressive degeneration of nigrostriatal dopamine neurons, the presence of inclusion bodies in dopamine neurons, and motor impairment. We examined in vitro and in vivo the effects of PSI on nigrostriatal dopamine neurons. Mass spectrometric analysis was employed to verify the authenticity of the PSI compound. PSI was non-specifically toxic to neurons in ventral mesencephalic organotypic slice cultures, indicating that impairment of proteasome function in vitro is toxic. Moreover, systemic administration of PSI transiently decreased brain proteasome activity. Systemic treatment of rats with PSI did not, however, result in any biochemical or anatomical evidence of lesions of nigrostriatal dopamine neurons, nor were any changes in locomotor activity observed. These data suggest that systemic administration of proteasome inhibitors to normal adult rats does not reliably cause an animal model of parkinsonism.

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