Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Intracellular localization of proteasomal degradation of a viral antigen.

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

The Human Genome Project: misguided science policy.

Genome mapping and sequencing projects are inappropriate and wasteful expenditures of precious research funds. By focusing on the acquisition of nucleotide sequences, the various genome projects emphasize the products of science over the process of science. It is doubtful that much of the resulting information will provide insights into human diseases or fundamental biological processes. The routine nature of genome sequencing makes it ill-suited for training young scientists. Such projects may also hamper the education of future investigators by diverting research support from universities to genome centers and commercial firms.

Circular pancreatic trypsin inhibitor. A novel substrate for studies on intracellular proteolysis.

Several recent studies indicate that substrates for ubiquitin-dependent proteolysis must possess unblocked alpha-amino termini. To examine further the importance of free amino groups for proteolytic susceptibility we selected pancreatic trypsin inhibitor (PTI) as a test substrate. PTI can be circularized to form cPTI, a molecule that lacks alpha-amino groups in the absence of an endoproteolytic cleavage. We compared the breakdown of radioiodinated PTI and cPTI in rabbit reticulocyte lysate and found that cPTI was not stabilized relative to PTI. In addition, proteolysis of PTI or cPTI was not inhibited upon conversion of their lysine residues to homoarginine. However, neither degradation of PTI nor cPTI required ATP, and ubiquitin conjugation to either molecule was minor relative to known substrates of the ubiquitin pathway. Thus, PTI and cPTI are cleaved by an ATP-independent endoprotease(s) that does not require the substrate to be ubiquitinated. Such an activity was identified in low salt fractions obtained upon DEAE chromatography of reticulocyte lysate. The ubiquitin/ATP-dependent protease and another large multisubunit protease, both of which elute from DEAE-Fractogel at higher salt concentrations, do not degrade PTI or cPTI. Although monomeric PTI was rapidly degraded in reticulocyte lysate, cross-linked PTI molecules were stable both in reticulocyte lysate and following introduction into cultured cells using red blood cell-mediated microinjection. Thus, increased rates of turnover do not necessarily correlate with greater molecular mass of the substrate.

Microinjection studies on selective protein degradation: relationships between stability, structure, and location.

We have used two approaches to study the relationship between the structure of a protein and its intracellular stability. First, over 35 proteins for which the primary and X-ray structure are known were radiolabeled and introduced into HeLa cells by RBC-mediated microinjection. We then measured the half-life and intracellular location of each protein. Analysis of these data has shown that the role of lysosomes in degradation of injected proteins is minor and nonselective, and that the subcellular location of a protein following injection may have a significant influence on its stability. Furthermore, no correlations were found between the half-life of a protein and its size, isoelectric point, hydrophobicity, or thermostability. However, initial multiple correlative analyses suggest that the stability of an injected protein may be related to an interplay between its location and the presence of unstable amino acids and disordered structure on the protein surface. Second, we have examined the primary sequences of 10 proteins with intracellular half-lives of less than 2 h. Each rapidly degraded protein contains at least one region of 12 to 45 amino acids rich in proline, glutamic acid, serine, and threonine (PEST). These PEST regions, flanked by positively charged amino acids, can also be identified by features common in their hydropathy plots. Similar inspection of 35 more stable, structurally characterized proteins revealed that only three contained weak PEST regions. On the basis of this information, we anticipated that caseins, which contain several PEST regions, would be rapidly degraded within eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation rates and intracellular distributions of structurally characterized proteins injected into HeLa cells.

The degradation rates of 35 proteins for which primary and X-ray structural data are available have been measured following their microinjection into HeLa cells. Each protein was radiolabeled by at least two techniques, and its degradation was measured in the presence or absence of the lysosomotropic agents, ammonia and chloroquine. The intracellular location of each protein was also determined by fractionation of injected cells using differential centrifugation in sucrose or by extraction in buffers containing Triton X-100. Preliminary analysis of these data indicates the following: lysosomes play a minor, nonselective role in the degradation of most microinjected proteins; the location of an injected protein within cells may significantly influence the rate at which it is degraded; and initial analyses of protein structural data such as molecular weight, isoelectric point, alpha or beta content, hydrophobicity, etc. have not yet revealed any strong correlations with stability.

Microinjection of thymidine kinase and bovine serum albumin into mammalian cells by fusion with red blood cells.

A procedure is described by which proteins can be rapidly and efficiently microinjected into large numbers of culture cells. Proteins were first introduced into mammalian red blood cells during hypotonic hemolysis, and the resealed red cells were subsequently fused to culture cells using Sendai virus. In seven experiments, thymidine kinase or 125I-BSA were transferred to culture cells during fusion. Although proteins were used in the present experiments, the microinjection procedure should work equally well for other macromolecules.