Biomedical subjects

M Bogyo

Publications and source records attributed to M Bogyo.

23 records · Page 2Linked to original sources

PubMed1998

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes.

BACKGROUND: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active beta subunits, little is known about the principles that govern substrate hydrolysis by the proteasome. RESULTS: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 125I-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of beta-subunit modification. CONCLUSIONS: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual beta subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual beta subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.

Amino Acid Sequence↗

PubMed1997

Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors.

The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a 125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus. 125I-NIP-L3VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.

ATP-Dependent Proteases↗

PubMed1997

Proteasome inhibitors and antigen presentation.

Protein degradation plays an important role in the control and regulation of many crucial biological functions, ranging from cell cycle progression to presentation of viral antigens for scrutiny by cells of the immune system. At the heart of many of these catabolic events is the multicatalytic proteinase complex known as the proteasome. This large barrel-shaped protein complex executes a remarkable set of functions ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling molecules. Inhibitors of this proteolytic complex have thus been extremely useful for perturbing its function and deciphering its role in these diverse biological processes. Inhibitors of the proteasome consist mainly of peptides that are modified at the predicted site of hydrolysis with a reactive functional group capable of modifying the attacking nucleophile, either reversibly or irreversibly. Many of these inhibitors can be used in living cells and have proved to be invaluable tools for the study of proteasome function.

Antigen Presentation↗

PubMed1996

Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.

The human cytomegalovirus genome encodes proteins that trigger destruction of newly synthesized major histocompatibility complex (MHC) class I molecules. The human cytomegalovirus gene US2 specifies a product capable of dislocating MHC class I molecules from the endoplasmic reticulum to the cytosol and delivering them to the proteasome. This process involves the Sec61 complex, in what appears to be a reversal of the reaction by which it translocates nascent chains into the endoplasmic reticulum.

Adenosine Triphosphate↗

PubMed1996

The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.

Human cytomegalovirus (HCMV) down-regulates expression of MHC class I products by selective proteolysis. A single HCMV gene, US11, which encodes an endoplasmic reticulum (ER) resident type-I transmembrane glycoprotein, is sufficient to cause this effect. In US11+cells, MHC class I molecules are core-glycosylated and therefore inserted into the ER. They are degraded with a half-time of less than 1 min. A full length breakdown intermediate that has lost the single N-linked glycan in an N-glycanase-catalyzed reaction transiently accumulates in cells exposed to the protease inhibitors LLnL, Cbz-LLL, and lactacystin, identifying the proteasome as a key protease. Subcellular fractionation experiments show this intermediate to be cytosolic. Thus, US11 dislocates newly synthesized class I molecules from the ER to the cytosol, where they are acted upon by an N-glycanase and the proteasome.

Astrocytoma↗