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Alfred L Goldberg

Publications and source records attributed to Alfred L Goldberg.

At least 37 records · Page 2Linked to original sources
PubMed2004

Nrdp1-mediated degradation of the gigantic IAP, BRUCE, is a novel pathway for triggering apoptosis.

Degradation of certain inhibitor of apoptosis proteins (IAPs) appears to be critical in the initiation of apoptosis, but the factors that regulate their degradation in mammalian cells are unknown. Nrdp1/FLRF is a RING finger-containing ubiquitin ligase that catalyzes degradation of the EGF receptor family member, ErbB3. We show here that Nrdp1 associates with BRUCE/apollon, a 530 kDa membrane-associated IAP, which contains a ubiquitin-carrier protein (E2) domain. In the presence of an exogenous E2, UbcH5c, purified Nrdp1 catalyzes BRUCE ubiquitination. In vivo, overexpression of Nrdp1 promotes ubiquitination and proteasomal degradation of BRUCE. In many cell types, apoptotic stimuli induce proteasomal degradation of BRUCE (but not of XIAP or c-IAP1), and decreasing Nrdp1 levels by RNA interference reduces this loss of BRUCE. Furthermore, decreasing BRUCE content by RNA interference or overexpression of Nrdp1 promotes apoptosis. Thus, BRUCE normally inhibits apoptosis, and Nrdp1 can be important in the initiation of apoptosis by catalyzing ubiquitination and degradation of BRUCE.

Apoptosis↗
PubMed2004

Post-proteasomal antigen processing for major histocompatibility complex class I presentation.

Peptides presented by major histocompatibility complex class I molecules are derived mainly from cytosolic oligopeptides generated by proteasomes during the degradation of intracellular proteins. Proteasomal cleavages generate the final C terminus of these epitopes. Although proteasomes may produce mature epitopes that are eight to ten residues in length, they more often generate N-extended precursors that are too long to bind to major histocompatibility complex class I molecules. Such precursors are trimmed in the cytosol or in the endoplasmic reticulum by aminopeptidases that generate the N terminus of the presented epitope. Peptidases can also destroy epitopes by trimming peptides to below the size needed for presentation. In the cytosol, endopeptidases, especially thimet oligopeptidase, and aminopeptidases degrade many proteasomal products, thereby limiting the supply of many antigenic peptides. Thus, the extent of antigen presentation depends on the balance between several proteolytic processes that may generate or destroy epitopes.

Animals↗
PubMed2004

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression.

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin-induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair-fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin-1/MAFbx and MuRF-1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down-regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up-regulated (lipin, metallothionein, AMP deaminase, RNA helicase-related protein, TG interacting factor) and several growth-related mRNAs were down-regulated (P311, JUN, IGF-1-BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.

Adenosine Triphosphate↗
PubMed2003

Protein degradation and protection against misfolded or damaged proteins.

The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

Humans↗
PubMed2003

The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites.

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.

Aldehydes↗
PubMed2003

The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation.

Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn't reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation.

Animals↗
PubMed2003

ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation.

To clarify the role of ATP in proteolysis, we studied archaeal 20S proteasomes and the PAN (proteasome-activating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S ATPases. PAN's ATPase activity was stimulated similarly by globular (GFPssrA) and unfolded (casein) substrates, and by the ssrA recognition peptide. Denaturation of GFPssrA did not accelerate its degradation or eliminate the requirement for PAN and ATP. During degradation of one molecule of globular or unfolded substrates, 300-400 ATP molecules were hydrolyzed. An N-terminal deletion in the 20S alpha subunits caused opening of the substrate-entry channel and rapid degradation of unfolded proteins without PAN; however, degradation of globular GFPssrA still required PAN's ATPase activity, even after PAN-catalyzed unfolding. Thus, substrate binding activates ATP hydrolysis, which promotes three processes: substrate unfolding, gate opening in the 20S, and protein translocation.

Adenosine Triphosphatases↗
PubMed2003

TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k.

In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

Animals↗
PubMed2002

An IFN-gamma-induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I-presented peptides.

Precursors to major histocompatibility complex (MHC) class I-presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residues from many antigenic precursors. It was identified as a metallopeptidase named "adipocyte-derived leucine" or "puromycin-insensitive leucine-specific" aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER-aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-gamma. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.

Animals↗
PubMed2002

The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues.

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) appears to be specialized to produce peptides presented on class I major histocompatibility complex molecules. We found that purified ERAP1 trimmed peptides that were ten residues or longer, but spared eight-residue peptides. In vivo, ERAP1 enhanced production of an eight-residue ovalbumin epitope from precursors extended on the NH2 terminus that were generated either in the ER or cytosol. Purified ERAP1 also trimmed nearly half the nine-residue peptides tested. By destroying such nine-residue peptides in normal human cells, ERAP1 reduced the overall supply of antigenic peptides. However, after interferon-gamma treatment, which causes proteasomes to produce more NH2-extended antigenic precursors, ERAP1 increased the supply of peptides for MHC class I antigen presentation.

Animals↗
PubMed2002

Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3.

The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases plays fundamental roles in the regulation of cell survival, proliferation, and differentiation. Here, we present evidence that ErbB3 is degraded by proteasomes, and that Nrdp1 (referred to as FLRF in mice) associates with ErbB3 and stimulates its ubiquitination and degradation by proteasomes. Nrdp1 mRNAs are expressed in a variety of human tissues. The N-terminal half of Nrdp1 possesses an atypical RING finger domain, which is required for enhancing ErbB3 degradation. Its C-terminal half by itself associates with ErbB3 and raises ErbB3 levels in cells, probably by acting as a dominant-negative form of Nrdp1. In cell-free systems, Nrdp1 has ubiquitin ligase (E3) activity and ubiquitinates ErbB3, as well as itself, in the presence of the ubiquitin-carrier protein (E2), UbcH5. These data indicate that Nrdp1 is a RING finger-type of ubiquitin ligase, which promotes degradation of ErbB3 by proteasomes and, thus, may be an important factor influencing cell growth.

Amino Acid Sequence↗
PubMed2002

Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures.

Trehalose accumulates dramatically in microorganisms during heat shock and osmotic stress and helps protect cells against thermal injury and oxygen radicals. Here we demonstrate an important role of this sugar in cold-adaptation of bacteria. A mutant Escherichia coli strain unable to produce trehalose died much faster than the wild type at 4 degrees C. Transformation of the mutant with the otsA/otsB genes, responsible for trehalose synthesis, restored trehalose content and cell viability at 4 degrees C. After temperature downshift from 37 degrees C to 16 degrees C ("cold shock"), trehalose levels in wild-type cells increased up to 8-fold. Although this accumulation of trehalose did not influence growth at 16 degrees C, it enhanced cell viability when the temperature fell further to 4 degrees C. Before the trehalose build-up, levels of mRNA encoding OtsA/OtsB increased markedly. This induction required the sigma factor, RpoS, but was independent of the major cold-shock protein, CspA. otsA/B mRNA was much more stable at 16 degrees C than at 37 degrees C and contained a "downstream box," characteristic of cold-inducible mRNAs. Thus, otsA/otsB induction and trehalose synthesis are activated during cold shock (as well as during heat shock) and play an important role in resistance of E. coli (and probably other organisms) to low temperatures.

Acclimatization↗
PubMed2002

Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes.

PA28 is a gamma-interferon-induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP-ubiquitin-dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with 'singly capped' 26S (i.e. 19S-20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no significant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28alphabeta or PA28alpha generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.

Amino Acid Sequence↗
PubMed2002

Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the alpha-rings.

The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.

Allosteric Site↗
PubMed2002

Protein degradation and the generation of MHC class I-presented peptides.

Over the past decade there has been considerable progress in understanding how MHC class I-presented peptides are generated. The emerging theme is that the immune system has not evolved its own specialized proteolytic mechanisms but instead utilizes the phylogenetically ancient catabolic pathways that continually turnover proteins in all cells. Three distinct proteolytic steps have now been defined in MHC class I antigen presentation. The first step is the degradation of proteins by the ubiquitin-proteasome pathway into oligopeptides that either are of the correct size for presentation or are extended on their amino-termini. In the second step, aminopeptidases trim N-extended precursors into peptides of the correct length to be presented on class I molecules. The third step involves the destruction of peptides by endo- and exopeptidases, which limits antigen presentation, but is important for preventing the accumulation of peptides and recycling them back to amino acids for protein synthesis or production of energy. The immune system has evolved several components that modify the activity of these ancient pathways in ways that enhance the generation of class I-presented peptides. These include catalytically active subunits of the proteasome, the PA28 proteasome activator, and leucine aminopeptidase, all of which are upregulated by interferon-gamma. In addition to these pathways that operate in all cells, dendritic cells and macrophages can also generate class I-presented peptides from proteins internalized from the extracellular fluids by degrading them in endocytic compartments or transferring them to the cyotosol for degradation by proteasomes.

Animals↗
PubMed2002

The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.

Three different proteolytic processes have been shown to be important in the generation of antigenic peptides displayed on MHC-class I molecules. The great majority of these peoptides are derived from oligopeptides produced during the degradation of intracellular proteins by the ubiquitin-proteasome pathway. Novel methods were developed to follow this process in vitro. When pure 26S proteasomes degrade the model substrate, ovalbumin, they produce the immunodominant peptide, SIINFEKL, occasionally, but more often an N-extended form of SIINFEKL. Interferon-gamma stimulates antigen presentation in part by inducing new forms of the proteasome that are more efficient in antigen presentation, and in vitro these immunoproteasomes specifically produce more of the N-extended versions of SIINFEKL. In addition, gamma-interferon induces a novel 26S complex containing the 19S and 20S particles and the proteasome activator, PA28, which we show cleaves proteins in distinct ways. In vivo studies established that proteasomal cleavages produce the C-termini of antigenic peptides, but not their N-termini, which can be formed efficiently by aminopeptidases that trim longer proteasomal products to the presented epitopes. gamma-interferon stimulates this trimming process by inducing in the cytosol leucine aminopeptidase and a novel aminopeptidase in the ER. Peptides released by proteasomes, including antigenic peptides, are labile in cytosolic extracts, and most of the longer proteasome products are rapidly cleaved by the cytosolic enzyme, thymet oligopeptidase (TOP). If cells express large amounts of TOP, class I presentation decreases, and if TOP is inhibited, presentation increases. Thus, peptide degradation in the cytosol appears to limit the efficiency of antigen presentation.

Amino Acids↗