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WO1994017816A1 - Role de la proteolyse dependant de l'ubiquitine atp dans la presentation de l'antigene restreint chm-1 et inhibiteurs utilises a cet effet - Google Patents

Role de la proteolyse dependant de l'ubiquitine atp dans la presentation de l'antigene restreint chm-1 et inhibiteurs utilises a cet effet Download PDF

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Publication number
WO1994017816A1
WO1994017816A1 PCT/US1994/001183 US9401183W WO9417816A1 WO 1994017816 A1 WO1994017816 A1 WO 1994017816A1 US 9401183 W US9401183 W US 9401183W WO 9417816 A1 WO9417816 A1 WO 9417816A1
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Prior art keywords
ubiquitin
inhibitor
cleavage
mhc
atp
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PCT/US1994/001183
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English (en)
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Alfred L. Goldberg
Kenneth L. Rock
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The President And Fellows Of Harvard College
Dana Farber Cancer Institute
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Application filed by The President And Fellows Of Harvard College, Dana Farber Cancer Institute filed Critical The President And Fellows Of Harvard College
Priority to EP94908690A priority Critical patent/EP0684829A4/fr
Priority to AU61691/94A priority patent/AU676721B2/en
Priority to JP6518184A priority patent/JPH08507754A/ja
Publication of WO1994017816A1 publication Critical patent/WO1994017816A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • autoimmune diseases There are many diseases that result from the immune system attacking and destroying tissues of the body. These conditions can cause severe morbidity and in many cases can be fatal. Some of these diseases, such as systemic lupus erythematosus, result from an individual's immune system reacting against its own cells as if they were foreign or pathogenic. Collectively these diseases are referred to as autoimmune diseases. Other diseases result from the immune system responding against molecules, such as therapeutic drugs, that are present in the body's tissues. Yet other diseases are caused when the immune system responds against transplanted foreign tissues in the body. The immune response against foreign tissues i ⁇ the major cause of failure of transplanted organs or grafts in transplantation and is the principle barrier to this procedure.
  • lymphocytes In these diseases, the cells that cause the destructive immune response are lymphocytes, and in the vast majority of cases, T lymphocytes play an obligatory role in these responses.
  • the immune response i ⁇ initiated when T lymphocytes recognize fragments of antigens that are bound to major histocompatibility complex (MHC) molecules on the surface of antigen- presenting cells.
  • MHC major histocompatibility complex
  • MHC molecules stimulates the T cells and directs them to respond against the cells bearing the antigen-MHC complex.
  • T lymphocytes There are several different kinds of T lymphocytes.
  • the two major subsets of T lymphocytes which are distinguished by the expression of certain cell surface molecules, differ in their specificity and function.
  • T cells that express the CD4 molecule are specific for antigens presented by MHC class II molecules and usually stimulate antibody or inflammatory responses.
  • T cells that express CD8 molecules are specific for antigens presented by MHC class I molecules and function to kill host cells.
  • the CD8 + T cells mediate tissue damage in several of the immune mediated diseases described above.
  • the therapeutic approaches to treating the immune- mediated diseases are directed at blocking the response of the lymphocytes.
  • cyclosporin A and steroids seek to block the response of lymphocytes by inhibiting lymphocyte activation.
  • Still other therapies such as treatment with monoclonal antibodies against cell interaction molecules, seek to block lymphocytes from interacting with other cells. All of these therapies have significant adverse side effects.
  • Several of the drugs have associated toxicities; e.g., cyclosporin is associated with nephrotoxicity, and steroids cause Cushing' ⁇ disease.
  • Monoclonal antibodies are associated with immune responses against the antibodies, which makes continued therapy ineffective. In all cases, immunosuppression results in increased susceptibility to infection, which can be lethal. A better approach to controlling or altering T cell responses would be very beneficial therapeutically.
  • cytolytic immune responses are useful for the treatment of a variety of autoimmune diseases and for preventing rejection of transplanted organs or grafts.
  • These methods and drugs block the generation of cellular immunity by preventing the initial presentation of cellular and viral antigens on MHC-class I molecules to T cells.
  • the methods comprise using inhibitors of the ATP-ubiquitin-dependent proteolytic pathway to prevent or reduce the processing of intracellular proteins into the antigenic peptides that bind to MHC-1 molecules.
  • This invention results from Applicants' work, which shows that inhibition of an early step (ubiquitin activation) and a late step (cleavage of intracellular proteins into peptides) in the ATP-ubiquitin-dependent proteolytic pathway can inhibit MHC-1 antigen presentation by blocking the generation of peptides binding to MHC-1 molecules.
  • This work provides strong evidence for the role of this cytosolic protein degradation system in the processing of MHC-1 presented antigens for MHC-1 presentation.
  • Applicants have discovered new features of the function of the proteoso e (multicatalytic protease complex) , an essential component of the ATP-ubiquitin pathway.
  • methods and drugs are provided for reducing cytolytic immune responses without affecting antibody-mediated or inflammatory responses.
  • Thi ⁇ invention should thus provide therapeutics useful for autoimmune diseases and prevention of transplant rejection without the attendant disadvantages of generalized immune suppression.
  • gamma interferon ⁇ -IFN
  • ⁇ -IFN gamma interferon
  • MHC-I antigen presentation can be blocked by inactivation of ubiquitin conjugation, an early step in the ATP- ubiquitin-dependent proteolytic pathway, It was found that, at the nonper issive temperature, clas ⁇ I- restricted antigen presentation was inhibited in a mutant cell line expressing a thermolabile ubiquitin activating enzyme (El) .
  • El thermolabile ubiquitin activating enzyme
  • chymostatin an inhibitor of chymotrypsin-like proteases, inhibits MHC-I presentation by antigen presenting cells.
  • Other inhibitor ⁇ particularly of the chymotrypsin-like and trypsin-like activities of proteasomes, are further described herein.
  • Figure 1 shows the effect of gamma interferon ( ⁇ - IFN) on peptidase activities of proteasomes from U937 cell ⁇ .
  • the upper panel shows the hydrolysis of the indicated substrates by proteasome fractions from control cells and cells treated with ⁇ -IFN.
  • the lower panel shows kinetic analysis of rates of cleavage of the sub ⁇ trates by the same proteasome fractions.
  • Figure 2 shows ⁇ the effect of ⁇ -IFN on peptidase activities of purified 20S and 26S proteasomes.
  • Figure 3 shows the difference in activities of the peptida ⁇ e ⁇ of protea ⁇ omes from wild-type and MHC- deficient lymphoblastoid cells.
  • the upper panel ⁇ how ⁇ the peptida ⁇ e activities.
  • the lower panel shows kinetic analysi ⁇ .
  • Figure 4 shows ⁇ the frequency of amino acid re ⁇ idues preceding bonds cleaved to generate peptides presented on MHC-cla ⁇ I molecule ⁇ .
  • the left panel shows the frequency of amino acids at the carboxyl-termini.
  • the right panel show ⁇ the frequency of amino acid ⁇ preceding the amino-terminal re ⁇ idue ⁇ .
  • Figure 5 shows MHC class I-restricted pre ⁇ entation of ovalbumin (OVA) by mutant (t ⁇ 20.10.2) and wild-type (E36.12.4) cell ⁇ .
  • Figure 6 (A-B) how ⁇ the effect of temperature on the synthesi ⁇ and maturation of H-2K b MHC heavy chain ⁇ .
  • Figure 7 (A-B) shows MHC clas ⁇ I-re ⁇ tricted pre ⁇ entation of endogenously synthesized OVA 257 _ 264 peptide.
  • Figure 8 shows inhibition of MHC clas ⁇ I-re ⁇ tricted pre ⁇ entation of ovalbumin (left) and ovalbumin peptide (right) by chy o ⁇ tatin.
  • This invention relate ⁇ to an approach for inhibiting cytolytic immune responses that avoids generalized ⁇ uppre ⁇ ion of T lymphocyte function with it ⁇ attendant risks of infection.
  • the methods and drugs of this approach are useful for treating autoimmune diseases and preventing rejection of foreign tissues, such as transplanted organs or grafts.
  • the strategy is to inhibit antigen presentation by major histocompatibility complex (MHC) I molecules rather than suppress T cell activity.
  • MHC major histocompatibility complex
  • This approach ha ⁇ the advantage that it selectively affects only clas ⁇ I MHC- restricted immune re ⁇ pon ⁇ e ⁇ and not antibody or other CD4 + T cell-mediated responses. Consequently, there should be less generalized immunosuppres ⁇ ion and susceptibility to infection in the patient.
  • Drugs that interfere with clas ⁇ I antigen presentation should also be useful in combination with other existing therapies and may lower the dose used in these therapies, which would decrease toxicities.
  • antigenic peptide ⁇ that binds to the MHC-I molecule ⁇ .
  • MHC-I binding peptides have strict sequence and size requirements.
  • the antigens are not presented at the cell surface and CD8 + T cells are not stimulated. This method ⁇ hould block both the initiation of immune re ⁇ ponse and stop ongoing immune response ⁇ .
  • Thi ⁇ invention is based on Applicants' work (see Examples below) supporting the role of the ATP-ubiquitin dependent proteolytic pathway, and in particular, protea ⁇ omes (multicatalytic proteinase complexe ⁇ ) , in the generation of MHC-I-a ⁇ sociated antigenic peptides.
  • An initial step in the presentation of intracellular and viral proteins to the immune system i ⁇ their proteolytic degradation in the cytosol to small peptides (Town ⁇ end and Bodmer, 1989) .
  • the antigenic fragments are then taken up via specific membrane transporters (Monaco, 1992; Powis, et al.
  • proteolytic ⁇ ystems exist in eukaryotic cells, and the ATP-ubiquitin pathway is the major cytosolic pathway of protein degradation (Finley and Chau, 1991; Hershko and Ciechanover, 1982; Rech ⁇ teiner, 1987) .
  • protein substrates are first modified by covalent conjugation to multiple ubiquitin moieties, which marks them for rapid degradation by a 26S (1,500 kD) ATP-dependent proteolytic complex, called the 26S proteo ⁇ o e or UCDEN (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; axman et al . , 1987; Hough et al .
  • This large structure contains the 20S (about 700 kD) proteasome as its proteolytic core plu ⁇ many additional regulatory and catalytic co ponent ⁇ (Goldberg and Rock, 1992; Orlow ⁇ ki, 1990; Rivett, 1989; Waxman et al ., 1987; Hough et al ., 1987; Armon et al . , 1990; Driscoll and Goldberg, 1990; Eytan et al . , 1989).
  • the precise intracellular function of the 20S proteosome is not clear; when i ⁇ olated, this particle can degrade proteins and oligopeptides, but not ubiquitin-conjugated proteins (Goldberg, 1992; Tanaka et al . , 1992; Armon, 1990; Driscoll and Goldberg, 1990a; Eytan et al . , 1989; Dri ⁇ coll and Goldberg, 1989) .
  • the 20S protea ⁇ ome is composed of about 15 distinct subunits of 20 - 30 kD. It contains three or four different neutral peptidases, which cleave specifically on the carboxyl side of hydrophobic, basic and acidic amino acid ⁇ (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; Orlowski, 1990; Rivett, 1989) . These peptidases are referred to as the chymotrypsin- like peptida ⁇ e, the trypsin-like peptidase, and the peptidylglutamyl peptidase, respectively.
  • the LMP particle ⁇ contain two polypeptide ⁇ , LMP 2 and LMP 7, which are encoded in the MHC chromosomal region.
  • ⁇ -IFN gamma interferon
  • Treatment of cell ⁇ with gamma interferon ( ⁇ -IFN) stimulates antigen presentation (Townsend and Bodmer, 1989; Yewdell and Bennink, 1992) and causes the induction of LMP 2 and LMP 7 , as well a ⁇ other MHC genes (Glynne et al . , 1991; Kelly et al . , 1991; Monaco and McDevitt, 1982; Yang et al .
  • LMP 2 and LMP 7 are two subunits of particles representing a small fraction of the 2OS proteosome population.
  • the importance of these subunit ⁇ in the immune re ⁇ ponse is uncertain, ⁇ ince deletion of the ⁇ e gene ⁇ does not prevent antigen presentation, in contrast to deletion of other MHC-gene ⁇ (Arnold et al . , 1992; Mamburg et al . , 1992) .
  • ⁇ -IFN can alter the polypeptide composition of 20S and 26S proteasome ⁇ (Yang et al. , 1992) in mutant ⁇ lacking the LMP 2 and 7.
  • thi ⁇ work described herein provides confirming evidence for a role for the 20S and 26S proteasome ⁇ and the ATP-ubiquitin proteolytic pathway in the generation of antigenic peptides for MHC-1 antigen pre ⁇ entation.
  • thi ⁇ work reveals new features of the pathway of proteolysis of antigens and of proteasome function, which led to the present invention. Firstly, they have shown that gamma interferon, which is known to ⁇ timulate antigen presentation, ⁇ electively enhances those peptidase activities, namely, the trypsin-like and chymotrypsin- like activities, of the proteasome ⁇ that produce peptide ⁇ that can bind to MHC-1 molecules, i.e.
  • Applicants have shown that a defect in ubiquitin conjugation, an early step in the ATP- ubiquitin-dependent proteolytic pathway, leads to reduced MHC-I-re ⁇ tricted antigen presentation (Example 2) .
  • OVA ovalbumin
  • the ⁇ e finding ⁇ provide evidence for the role of the ATP-ubiquitin proteolytic ⁇ y ⁇ tem in MHC-I antigen pre ⁇ entation. They de on ⁇ trate that a block at either an early ⁇ tep (ubiquitin conjugation) or a late ⁇ tep (proce ⁇ ing of carboxyl-termini of the peptide ⁇ ) in the proteoly ⁇ i ⁇ of intracellular protein ⁇ by the ATP- ubiquitin system can inhibit clas ⁇ I restricted presentation. It is reasonable to expect that inhibition of other steps in this proteolytic pathway will al ⁇ o produce a ⁇ i ilar effect.
  • the inhibitor ⁇ can be naturally i ⁇ olated or synthetic and peptide or non-peptide molecules.
  • the inhibitors would selectively inhibit the chymotrypsin-like and tryp ⁇ in-like peptida ⁇ e ⁇ , while leaving the peptidylglutamyl peptida ⁇ e relatively unaffected.
  • a typical inhibitor would be a peptide aldehyde, like leupeptin, which inhibit ⁇ primarily cleavage after ba ⁇ ic residues, or chymostatin, which inhibit ⁇ primarily after hydrophobic residues.
  • Novel molecule ⁇ can also be obtained and tested for inhibitory activity.
  • various strategies are known in the art for obtaining the inhibitors for a given protease.
  • Compound or extract libraries can be screened for inhibitors using peptidase assays.
  • peptide and peptidomimetic molecules can be designed based on knowledge of the substrates of the protease.
  • substrate analogs can be synthesized containing a reactive group likely to interact with the catalytic site of the protease (see, e.g., Siman et al . , WO 91/13904; Powers et al . , 1986).
  • the inhibitors can be stable analogs of tran ⁇ ition intermediate ⁇ (tran ⁇ ition state analogs), such as Cbz-Gly-Gly-leucinal, which inhibits the chymotrypsin-like activity of the proteosome (Orlowski, 1990; see also Kennedy and Schultz, 1979) .
  • variant ⁇ or analogs of known inhibitor ⁇ , such as chymostatin can be also be ⁇ ynthesized.
  • protea ⁇ e inhibitor ⁇ reported in the literature, or molecule ⁇ ⁇ imilar to them, are likely to inhibit the activity of the two protea ⁇ omal peptidases. These include peptides containing an ⁇ -dicarbonyl unit, such a ⁇ an ⁇ -diketone or an ⁇ -keto ester, peptide chloromethyl ketones, isocou arins, peptide sulfonyl fluorides, peptidyl boronates, peptide epoxides and peptidyl diazomethanes (Angela ⁇ tro et al . , 1990; Bey et al . , EPO 363,284; Bey et al .
  • Ubiquitin conjugation itself i ⁇ a multistep process, involving the activities of an ubiquitin activating enzyme (El) , a group of ubiquitin-carrier protein ⁇ (E2) , which catalyze the tran ⁇ fer of ubiquitin to target protein ⁇ , and the ubiquitin-protein liga ⁇ e, E3.
  • Molecule ⁇ that block ubiquitin conjugation at any of these ⁇ tep ⁇ are al ⁇ o expected to be inhibitor ⁇ of antigen pre ⁇ entation.
  • ⁇ pecific inhibitors of these enzymes will be used, such as ⁇ ub ⁇ trate and tran ⁇ ition state analogs.
  • adeno ⁇ yl- pho ⁇ pho-ubiquitinol an analog of the sub ⁇ trate ubiquitin adenylate, has been found to be a ⁇ pecific and effective inhibitor of El (Wilkinson et al . , 1990).
  • the inhibitors can be u ⁇ ed in vitro or in vivo to block MHC-I antigen pre ⁇ entation. They can be admini ⁇ tered by any number of known routes, including orally, intravenously, intramu ⁇ cularly, topically, and by infu ⁇ ion (see, e.g., Platt and Stracher, U.S. Patent 4,510,130; Badalêt et al . , 1989; Staubli et al . , 1988).
  • the inhibitors are low molecular weight molecules. Suitable vehicles for protein drug delivery, such as liposomes, may al ⁇ o be used. Vehicles than can target the drug to ⁇ pecific tissues can also be u ⁇ ed. Examples
  • the present ⁇ tudie ⁇ were undertaken to clarify the role of ⁇ -IFN, proteasome ⁇ , and MHC-encoded protein ⁇ in the proce ⁇ ing of cell antigen ⁇ .
  • the work de ⁇ cribed below demon ⁇ trate ⁇ that the pattern of peptida ⁇ e activitie ⁇ of the 20S and 26S proteasome complexes i ⁇ altered following treatment of cells with ⁇ -IFN and upon expression of the MHC region that contains the LMP 2 and 7 genes.
  • These changes in catalytic activities should favor the generation of the types of peptide sequences (Falk et al . , 1991) that bind specifically to MHC-cla ⁇ I proteins.
  • protea ⁇ omes are found either as part of the 26S (1,500 kD) complexes, which degrade preferentially ubiguitin-conjugated proteins, or as 20S particles, which by themselves cannot hydrolyze Ub- conjugated proteins, despite having multiple proteolytic activitie ⁇ (Goldberg and Rock, 1992; Goldberg, 1992; Tanaka et al . , 1992; Armon et al . , 1990; Dri ⁇ coll and Goldberg, 1992; Eytan et al . , 1989; Dri ⁇ coll and Goldberg, 1989; Matthews et al . , 1989) .
  • Proteasomes from the mutant cells were found to degrade the basic sub ⁇ trate at approximately 40% of the rate and the hydrophobic sub ⁇ trate at 70% of the rate of the wild-type cell ⁇ ( Figure 3) .
  • proteasome ⁇ from the MHC-deleted variant con ⁇ istently cleaved the acidic substrate, Cbz-LLE-MNA, about 40% faster than similar preparations from wild-type cell ⁇ ( Figure 3) .
  • the change ⁇ in the relative activitie ⁇ of the three peptidases should alter the nature of the oligopeptides that are generated by the protea ⁇ ome and are available for further proteolytic proce ⁇ ing and/or delivery to MHC-class I molecules (see below) .
  • ⁇ -IFN is known to stimulate the expres ⁇ ion of the two MHC-encoded subunits, LMP 2 and LMP 7, and to increase the abundance of "LMP particles" in cells (i.e. those proteasomes containing LMP subunits) .
  • the simplest explanation for these finding ⁇ is that the ⁇ -IFN-induced alterations in catalytic properties result from incorporation into proteasomes of these MHC-encoded subunits.
  • the LMP 2 and/or LMP 7 proteins may themselves contain "tryptic- like” or "chymotryptic-like" sites. If so, these sites may supplement the peptidase sites present in the absence of ⁇ -IFN or may replace them with sites of a higher catalytic efficiency.
  • ⁇ -IFN clearly induce some change ⁇ in protea ⁇ ome activity in the mutant ⁇ lacking LMP 2 and LMP 7 gene ⁇ .
  • ⁇ -IFN caused a small (but reproducible) increase in the chymotryptic activity.
  • the peptidylglutamyl activity in the mutants increases upon ⁇ -IFN treatment, even though in wild-type cells, this activity decreases.
  • ⁇ -IFN can reduce or suppress the synthesis of subunits encoded outside of this portion of the MHC region or may otherwise modify proteasome structure.
  • the LMP 2- and LMP 7-containing populations probably differ to a very large degree from the preexistent population in their hydrolytic activities, since the induction of the ⁇ e sub ⁇ et ⁇ or protea ⁇ omes by ⁇ -IFN were observed to cause large changes in the total peptida ⁇ e activitie ⁇ in the cell within 3 day ⁇ .
  • the changes in catalytic activity found here probably undere ⁇ timate the actual difference ⁇ between the activitie ⁇ of LMP-containing and LMP-deficient ⁇ ub ⁇ et ⁇ of protea ⁇ ome ⁇ .
  • proteasome propertie ⁇ In the pa ⁇ t, the proteasome has generally viewed as a single type of structure with characteristic enzymatic propertie ⁇ . This view is clearly simplistic or incorrect in light of the present findings, which emphasize the functional heterogeneity and plasticity of these particles.
  • protea ⁇ ome propertie ⁇ change in vivo during infection ⁇ or ⁇ ep ⁇ i ⁇ or other conditions, in which ⁇ -IFN level ⁇ ri ⁇ e and immune re ⁇ ponses are activated.
  • the ⁇ e alterations in proteasome propertie ⁇ must depend al ⁇ o on dosage and duration of exposure to thi ⁇ cytokine.
  • the existence of functionally distinct subclasses of proteasome ⁇ in cells may also be important in many other biological contexts.
  • the changes in the proteaso al activitie ⁇ induced by ⁇ -IFN or re ⁇ ulting from the MHC- deletion should alter the peptides generated by the proteasome during degradation of cellular and viral proteins, and thus change the repertoire of peptides available for presentation to T cells.
  • chymotryptic-like and “tryptic-like” activities and the decrease in peptidylglutamyl activity should generate more short peptides ending with basic or hydrophobic carboxyl termini, and fewer peptides ending with acidic carboxyl termini.
  • Applicants have found that the ⁇ -IFN-treatment and MHC-deletion change the peptida ⁇ e activities of proteasomes in the soluble fraction of the cell, as well as the activities of the minor population of proteasomes associated with ER (10% total) and with the nuclear fraction. For transport into the ER, these antigenic peptides generated by soluble proteasomes must somehow withstand complete hydrolysis by cytosolic exopeptidases.
  • U937 cells (0.15 X 10 6 /ml) were grown in 150 cm 3 flasks, in RPMI 1640 medium containing 10% FCS and antibiotics at 37°C for 72 hours in the presence or absence of 1000 U/ml of human recombinant ⁇ -IFN (kindly provided by Biogen, Inc. Cambridge, MA) . In preliminary studies, this concentration of ⁇ -IFN was found to cau ⁇ e maximal change ⁇ of proteasomal peptidase ⁇ activitie ⁇ in U937 cells. Mutant (721.174) and wild-type (721) cell ⁇ were grown for three days in the presence or absence of 3,000 U/ml of ⁇ -IFN, which was found to cau ⁇ e maximal change ⁇ in peptida ⁇ es activities.
  • Protea ⁇ ome fractions or crude extracts (0.1 mg/ml) and purified proteasomes (5 ⁇ g/ml of 20S or 15 ⁇ g/ml of 20S) ; were incubated in the Buffer B (see Detailed Description of Figure 1) with radioactive substrates at 37°C for 60 or 120 minutes in the presence of 2 mM ATP or 5 U/ml of apyrase (to de ⁇ troy residual ATP) .
  • Nonradioactive lactalbumin (20 ⁇ g/ml) was added to reaction mixtures containing Ub- 125 I-lactalbumin.
  • cell ⁇ were collected by centrifugation for 5 minute ⁇ at 700 x g, wa ⁇ hed twice, and re ⁇ uspended in Homogenization Buffer (50 mM Tris, 5 M MgCl 2 , 1 mM dithiothreitol (DTT), 2 mM ATP, 250 mM sucrose, pH 7.4).
  • Homogenization Buffer 50 mM Tris, 5 M MgCl 2 , 1 mM dithiothreitol (DTT), 2 mM ATP, 250 mM sucrose, pH 7.4
  • Cell suspensions were homogenized by several passages through a Dounce homogenizer (Wheaton) , followed by vortexing for 3 minutes with glass beads. After centrifugation at 10,000 x g for 20 minutes, the supernatant (“crude extract”) was centrifuged for 1 hour at 100,000 x g to obtain a "microso e pellet”.
  • the "proteasome fraction” was obtained by spinning the post- microsomal supernatant for 5 hours at 100,000 x g. Pellets were resuspended in about 10 volumes of Buffer A (50 mM Tris, 5 mM MgCl 2 , 1 mM DTT, 2 mM ATP, 20% glycerol), homogenized and centrifuged at 14,000 rpm for 15 minutes. The supernatant ⁇ , which contained 20% of the protein in crude extract ⁇ , were adju ⁇ ted to a concentration of 1 mg/ml (Bradford, 1976) prior to assay of proteolytic activities, as were the crude extract ⁇ (after dialysis against Buffer A) . With each set of cultured cells, two sets of extracts were prepared and analyzed in parallel. The averages of results obtained with the two sets were taken as the result of a particular experiment.
  • Buffer A 50 mM Tris, 5 mM MgCl 2 , 1 mM DTT, 2 mM ATP,
  • the upper panel of Figure 1 ⁇ hows the hydrolysis of the indicated substrates (100 ⁇ M) by proteasome fractions from control cells and cell ⁇ treated with ⁇ - IFN (Mamburg et al . , 1992) .
  • Peptidase activities were determined by the release of 7-amino-4-methylcoumarin (MCA) or methoxynaphthylamine (MNA) from fluorogenic peptide substrate ⁇ (I ⁇ chiura et al . , 1985) .
  • V ma ⁇ for Suc- LLVY-MCA was 0.7 units and 1.6 with ⁇ -IFN; for Boc-LRR- MCA, 0.3 units and 1.9 with ⁇ -IFN; for Cbz-LLE-MNA, 0.3 units and 0.2 with ⁇ -IFN.
  • K-, for Suc-LLVY-MCA was 0.2 mM and 0.3 M with ⁇ -IFN; for Boc-LRR-MCA, 0.4 mM and 0.5 mM with ⁇ -IFN; for Cbz-LLE-MNA, 0.10 mM and 0.06 mM with ⁇ -IFN.
  • Figure 2 shows the effect of ⁇ -IFN treatment on peptidase activities in 20S and 26S proteasomes purified from U937 cells. The results are representative of 3 different experiments. All difference ⁇ between control preparation ⁇ and tho ⁇ e from ⁇ -IFN-treated cells were statistically significant (P ⁇ 0.01), but no significant difference was seen for Cbz-LLE-MNA cleavage by 2OS. Units and assay conditions are as in Figure 1, but the final concentrations of the protea ⁇ ome ⁇ were 2.5-3.0 ⁇ g/ml (26S) or 8-10 ⁇ g/ml (20S) .
  • 20S and 26S proteasomes were isolated from the protea ⁇ ome fraction by Q Sepharose anion exchange and Superose 6 gel filtration FPLC chromatographies (Pharmacia) (Driscoll and Goldberg, 1989; Matthews et al . , 1989).
  • the 26S- rich fraction was identified by its ability to support the ATP-dependent degradation of Ub- 125 I-lactalbumin. Routinely, about 30 ⁇ g of 26S and 90 ⁇ g of 20S were obtained from 5 mg of proteasome fraction.
  • the upper panel of Figure 3 show ⁇ the difference in activities of the peptidases of proteasomes from wild- type (721) and MHC-deficient (721.174) lymphoblastoid cells.
  • the lower panel of Figure 3 shows the kinetic analysis of cleavage for all the sub ⁇ trates at different concentrations (S) by the same proteasome fractions from wild-type and mutant cells.
  • V ma ⁇ for Suc-LLVY-MCA was 6.5 unit ⁇ (wild-type) versus 2.4 (mutant) ; for Boc-LRR- MCA, 1.6 unit ⁇ v ⁇ 0.7; for Cbz-LLE-MNA, 0.7 units v ⁇ 1.2.
  • the left panel of Figure 4 shows the frequency of amino acid ⁇ at the carboxyl termini of 44 peptide ⁇ that bind MHC cla ⁇ s I molecules.
  • the data were collected from published sequence ⁇ of peptides (Falk et al . , 1991; Guo et al . , 1992; Hunt et al . , 1992; Jardetzky et al . , 1991; Mat ⁇ umura et al . , 1992).
  • the ⁇ e sequences were determined in different studies either by Edman degradation of individual peptide ⁇ that were eluted and purified from MHC cla ⁇ I molecule ⁇ or by functional analysis of synthetic antigenic peptides and alignment to known MHC-binding motifs.
  • the MHC class I molecules included in this analysis fall into two groups: K b , D b , K d , L d and HLA-A2.1, which bind peptides that generally have a hydrophobic C-terminal re ⁇ idue, and HLA-B27 and HLA-A 68, which bind peptide ⁇ that generally have a basic C-terminal residue.
  • the right panel of Figure 4 shows the frequency of amino acids preceding the N-termini of MHC class I-bound peptide ⁇ .
  • the protein sequences from which 39 of the peptides in the left panel were derived were available. Data were collected from the EMBL (Heidelberg, Germany) and National Biomedical Research Foundation (Bethesda, Md) database ⁇ .
  • T ⁇ 20 cell ⁇ a chemically mutagenized variant of the Chine ⁇ e ham ⁇ ter lung cell line E36, express a thermolabile ubiquitin-conjugating enzyme El (Kulka et al . , 1988).
  • El catalyzes the ATP-dependent activation of ubiquitin, an essential first step in conjugation of ubiquitin to cellular proteins (Hershko and Ciechanover, 1992; Irelandsteiner, 1987).
  • Ubiquitin-protein conjugation and protein degradation are reduced in ts20 cells at nonpermissive temperature (41°C) (Kulka et al . , 1988; Gropper et al . , 1991).
  • El is irreversibly inactivated in ts20 cells at 41°C.
  • ts20 and E36 cells were transfected with the cDNA gene ⁇ for H-2K b and ICAM-1 to enable the cell ⁇ to pre ⁇ ent Ag to the OVA- ⁇ pecific, K b -re ⁇ tricted murine T-cell hybridoma line RF33.70 (Rock et al . , 1990) .
  • the tran ⁇ fected cell line ⁇ , ts20.10.2 (mutant) and E36.12.4 (wild-type) were incubated at 41°C or 37°C for 1 hour before introducing OVA into the cytosol by osmotic lysi ⁇ of pino ⁇ ome ⁇ (Town ⁇ end et al .
  • Mutant cell ⁇ incubated at 41°C or 37°C did not differ in their uptake of extracellular fluid (0.94 ⁇ 0.27 nl/10 6 cell ⁇ at 41°C ver ⁇ u ⁇ 0.96 ⁇ 0.47 nl/10 6 cell ⁇ at 37°C, mean ⁇ SD of four experiment ⁇ ) as determined by the uptake of poly(vinylpyrrolidone) (PVP) , a commonly used marker of fluid-phase pinocytosi ⁇ (Wiley and McKinley, 1987) .
  • PVP poly(vinylpyrrolidone)
  • mutant cell ⁇ incubated at 41°C to present cyto ⁇ olic OVA could have been due to a block in the generation or presentation of the antigenic OVA peptide-K b complexes.
  • Incubation at 41°C did not affect the ability of mutant ( Figure 5C) or wild-type ( Figure 5D) cells to present exogenou ⁇ 0VA 257 - 264 peptide (amino acid ⁇ 257-264 of ovalbumin) added to the medium.
  • the ⁇ ynthetic 0VA 257 _ 264 peptide which repre ⁇ ent ⁇ the naturally proce ⁇ ed epitope from ovalbumin
  • binds directly to K b on the cell surface (Falk et al .
  • H-2K b molecules were sequentially immunoprecipitated with a mAb (Y-3) (Townsend et al . , 1990) that is specific for assembled K b molecule ⁇ . This wa ⁇ followed by precipitation with rabbit anti-heavy chain antisera (exon 8) (Smith and Barber, 1990) . Similar amounts of as ⁇ embled H-2K b were immunoprecipitated from mutant cell ⁇ incubated at the two temperature ⁇ ( Figure 6A) . Applicant ⁇ also found at both temperatures similar amounts of free heavy chains ( Figure 6A) and B 2 -microglobulin that were potentially available for a ⁇ sembly with peptide.
  • mutant cells treated as described in Figure 5, were incubated with OVA or infected with a recombinant vaccinia virus (Ova 257 _ 264 Vac) containing a minigene encoding the 0VA 257 _ 26 peptide preceded by an initiating Met.
  • OVA recombinant vaccinia virus
  • the immune system utilizes the cell's two primary and phylogenetically old degradative pathways to supply antigenic peptides: the ATP-ubiquitin-proteasome dependent pathway for clas ⁇ I and the vacuolar pathway for class II-restricted pre ⁇ entation.
  • FIG. 5 Show ⁇ MHC cla ⁇ I-restricted presentation of OVA by mutant (ts20.10.2) and wild-type (E36.12.4) cell ⁇ .
  • Ts20.10.2 (A and C) and E36.12.4 (B and D) cells were incubated at 41°C (filled) or 37°C (open) and then incubated with (A and B) or without (C and D) OVA.
  • the indicated number of APCs incubated with OVA were cultured with RF33.70 (A and B) , or control APC ⁇ (5 x IO 4 ) were cultured with RF33.70 and the indicated concentration of 0VA 257 _ 264 peptide (C and D) .
  • 12.4 (wild-type) cells (2 x IO 6 cells/ml) were incubated for 1 hour at 41°C or 37°C.
  • the cells were incubated for 10 minutes at 37 °c with 20 mg/ml OVA in hypertonic media, treated with hypotonic media, and then washed with ice-cold, serum-free RPMI 1640, as previously described (Townsend et aJ . , 1986; Michalek et al . , 1991) .
  • the washed APCs (2 x 10 6 cells/ml) were incubated for 1 hour at 37°C to allow for the expression of processed Ag on the cell surface.
  • the H-2K b cDNA, pBG367-K b was kindly provided by Dr. Gerald aneck.
  • the vector pcDL-SRccr296 was provided by Dr. Naoko Arai (Takebe et al . , 1988).
  • the H-2K b cDNA was subcloned into the Xhol/BamHI sites of the pcDL- SR ⁇ 296 vector.
  • Murine ICAM-I cDNA in the pH B APr-l-neo vector was provided by Drs. Hedrick and Brian (Siu et al . , 1989) .
  • E36 and t ⁇ 20 cells were cotran ⁇ fected with H-2K b and ICAM-I using the Lipofectin reagent (BRL) and ⁇ election with G418 (GIBCO) were performed as previously described (Dang et al . , 1990) , except ts20 cells were grown at 37 °C following trans ection.
  • G418-resi ⁇ tant clone ⁇ were screened for expression of ICAM-I and H2K b with mAb YNl/1.7.4 (Dang et al . , 1990) and B8-24-3 (Kohler et al . , 1981), respectively.
  • the transfected cell lines ts20.10.2 and E36.12.4 were pas ⁇ aged at 31°C and 37°C, respectively.
  • Figure 6 show ⁇ the effect of temperature on the synthesis and maturation of H-2K b .
  • mutant (ts20.10.2) and wild-type (E36.12.4) cell ⁇ were incubated for 1 hour at 41°C or 37°C and then metabolically radiolabeled for 15 minutes at 37°C.
  • H-2K b was sequentially immunoprecipitated with Y-3 (Jones and Janeway, 1981) followed by precipitation with anti-exon 8 (Smith and Barber, 1990) (a kind gift from Dr. Brian Barber, University of Toronto) and analyzed by one-dimensional SDS-PAGE.
  • H-2K b heavy chains (H) and B 2 -microglobulin (L) and of the relative molecular as ⁇ standards (M r x IO- 3 ) are indicated in Figure 6.
  • H-2K b wa ⁇ immunoprecipitated with Y-3 and analyzed by two-dimen ⁇ ional IEF/SDS-PAGE. Immunoprecipitate ⁇ made at the 0 time point of the cha ⁇ e were analyzed and showed a single immature heavy chain spot, which chased into the more acidic and larger molecular ma ⁇ forms shown above.
  • the heavy chains of H-2K b are indicated by the H, and the B 2 -microglobulin is indicated by the L.
  • Ts20.10.2 cells were incubated at 41°C (filled) or 37°C (open) for 1 hour and then either infected with
  • Ts20.10.2 cell ⁇ (7 x IO 5 cells/ml) were incubated for 1 hour at 41°C or 37°C and then infected with recombinant vaccinia viru ⁇ (10 PFU/cell) for 1 hour at 37°C ( Figure 7A) .
  • Ts20.]0.2 cells were incubated at 41°C and 37°C, treated with OVA, and then incubated at 37°C as described above (see Detailed Description of Figure 5) .
  • the APCs infected with vaccinia virus or incubated with OVA were fixed with 1% paraformaldehyde and incubated in microcultures with RF33.70 (IO 5 cell ⁇ ) as described in the Detailed Description of Figure 5.
  • microcultures were prepared, incubated, and as ⁇ ayed for IL-2 content a ⁇ also described therein.
  • APCs infected with a recombinant vaccinia virus containing an influenza nucleoprotein gene did not stimulate RF33.70.
  • Time-cour ⁇ e experiments with Ova 257 _ 26 ac showed that the amount of 0VA 257 _ 26 peptide-K b complexes on the surface of cells infected for 1 hour was limiting because ceil ⁇ infected for 3 hours were 64-fold more efficient at stimulating RF33.70.
  • Ova 25 _ 264 Vac was constructed by inserting a synthetic oligonucleotide behind the vaccinia virus p7.5 early/late promoter in pScll (Chakrabati et al . , 1985), which was modified such that the restriction sites Sail and iVotl were substituted for the Smal site.
  • the antigen-presenting cell ⁇ (APC ⁇ ) in this experiment were a mouse B lymphoblastoid cell line
  • the LB27.4 cells were washed serum free and incubated in the presence or absence of chymostatin (200 ⁇ g/ml; Bo ⁇ hringer Mannheim, Indianapoli ⁇ , IN) for one hour at 37°C. The cells were then resu ⁇ pended in
  • Electroporation buffer pho ⁇ phate buffered ⁇ aline, 1 mM Hepe ⁇ , 0.4 M mannitol; chymo ⁇ tatin was also added to the appropriate group ⁇
  • the resuspended cells were then subjected to electroporation on the CELL-PORATOR from Gibco BRL (Gaither ⁇ burg, MD) at 4°C with ⁇ etting of high ohms, capacitance 1180.
  • the cells were washed 4 times at 4°C and incubated at 37°C for two hour ⁇ in the continued presence or absence of chymostatin. After this two hour incubation, the cell ⁇ were fixed with 1% paraformaldehyde for 10 minutes at room temperature, followed by washing.
  • the indicated number of fixed antigen-presenting cells were incubated with 10 5 RF33.70 cells (an OVA + K b ⁇ pecific T-T hybridoma) in duplicate 200 ⁇ l microcultures. After 18 hours at 37°C, an aliquot (100 ⁇ l) of culture supernatant was harvested and a ⁇ ayed for IL-2 content using HT-2 cells, as described in Rock et al . , 1990a and 1990b.
  • Figure 8 shows MHC-I presentation of ovalbumin (OVA left panel) and an ovalbumin peptide (right panel) .
  • APCs were treated with (open circles) or without (closed circles) chymo ⁇ tatin and electroporated with ovalbumin.
  • APCs were treated with (open circles) or without (closed circles) chymostatin and electroporated with ovalbumin peptide.
  • the left panel demonstrates that chymostatin inhibits the presentation of ovalbumin with class I MHC molecules.
  • the right panel demonstrates that chymostatin does not inhibit the presentation of electroporated peptide.
  • a 40 kDa polypeptide regulator of the proteasome which inhibits the proteasome's proteolytic activities, has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis.
  • the isolated inhibitor exists as a 250 kDatuber and is quite labile (at 42°C) . It can be stabilized by the addition of ATP or a non-hydrolyzable ATP analog, although the purified inhibitor does not require ATP to inhibit proteasome function and lacks ATPase activity.
  • the inhibitor has been ⁇ hown to corre ⁇ pond to an e ⁇ ential component of the 1500 kDa proteolytic complex.
  • reticulocyte ⁇ are depleted of ATP, the 1500 kDa UCDEN is not found. Instead, Ganoth et al . identified three components, designated CF-1, CF-2 and CF-3, (J. Biol . Chem . 263:12412-12419 (1988)). The inhibitor isolated as described herein appears identical to CF-2 by many criteria. These findings indicate the idea that the inhibitor plays a role in the ATP-dependent mechanism of the UCDEN complex. It is possible, for example, that during protein breakdown, within the 1500 kDa complex, ATP hydrolysis leads to functional release of the 40 kDa inhibitor, temporarily allowing proteasome activity, and that ubiquitinated proteins trigger this mechanism.
  • the purified factor has been shown to inhibit hydrolysis by the proteasome of both a fluorogenic tetrapeptide and protein ⁇ ub ⁇ trate ⁇ .
  • the proteasome and partially purified CF-l were mixed in the presence of ATP and Mg + , the 1500 kDa complex was reconstituted and degradation of Ub- 125 I- lysozyme occurred.
  • DEAE-cellulose (DE-52) , CM-cellulose (CM-52), and phosphocellulo ⁇ e (Pll) were obtained from Whatman.
  • Ub- conjugating enzyme ⁇ (El, E2 and E3) were i ⁇ olated using Ub-sepharose affinity column chromatography (Hershko et al . , J . Biol . Chem . 258:8206-8215 (1983)), and were used to prepare Ub- 125 I-lysozyme conjugates (Hershko and Heller, Biochem . Biophys . Res . Comm . 128:1079-1086 (1985)) . All other materials used were as de ⁇ cribed in the previou ⁇ exa ple ⁇ .
  • Rabbit reticulocyte ⁇ induced by phenylhydrazine injection were prepared (as described previously or purcha ⁇ ed from Green Hectare ⁇ (Oregon, WI) . They were depleted of ATP by incubation with 2, 4-dir.” trop ' .-enol and 2-deoxyglucose as described (Ciechanover et al . , Biochem . Biophys . Res . Comm . 81:1100-1105 (1978)) . Ly ⁇ ates were then prepared and subjected to DE-52 chromatography. The protein eluted with 0.5M KC1 (Hershko et al . , J . Biol . Che .
  • the precipitated material was collected as above and then suspended in Buffer A containing 10% glycerol. After dialysis against this buffer, the 0-38% pellet was chromatographed on a Mono-Q anion exchange column equilibrated with Buffer A containing 10% glycerol. The protein was eluted using a 60 ml linear NaCl gradient from 20 to 400 mM. Fractions which inhibited the peptidase activity of the proteasome were pooled, concentrated, and then chromatographed on a Superose 6 (HR 10/30) gel filtration column equilibrated in Buffer A containing 100 mM NaCl and 0.2 mM ATP.
  • the column was run at a flow rate of 0.2 ml/minute, and 1 ml fraction ⁇ were collected. Further purification of the inhibitor wa ⁇ achieved by a second more narrow Mono-Q chromatographic gradient (from 50 to 300 mM NaCl) , which yielded a sharp peak of inhibitor where only the 40 kDa band was vi ⁇ ible after SDS-PAGE and Cooma ⁇ ie ⁇ taining. Fractions with inhibitory activity against the protea ⁇ ome were pooled and dialyzed against Buffer B which contained 20 mM KH 2 P0 4 (pH 6.5) , 10% glycerol, 1 mM DTT and 1 mM ATP.
  • the sample was then applied to a 2 ml pho ⁇ phocellulose column equilibrated in Buffer B.
  • the column was washed with 4 ml of this buffer, followed by 4 ml of this buffer, followed by 4 ml of Buffer B containing either 20, 50, 100, 400 or 600 mM NaCl.
  • the Mono-Q fractions that eluted from 100 to 240 mM NaCl were pooled, concentrated to 1 ml and applied to a supero ⁇ e 6 column equilibrated in Buffer A containing 100 mM NaCl and 0.2 mM ATP. The fractions eluting at approximately 600 kDa were used as the CF-1 containing fraction.
  • the proteasome was isolated from the supernatants of the two 38% ammonium sulfate precipitations. The supernatants were brought to 80% saturation with ammonium sulfate and mixed for 20 minutes. The precipitated protein was collected by centrifugation, resuspended in Buffer A, and dialyzed extensively against this buffer. The proteasome was isolated by Mono-Q anion exchange chromatography followed by gel filtration on supero ⁇ e 6 as described previously (Dri ⁇ coll and Goldberg, Proc . Natl . Acad . Sci . , USA 86:789-791 (1989)).
  • Tiie 1,500 kDa proteolytic complex wa ⁇ generated by incubating reticulocyte fraction II at 37°C for 30 minute ⁇ in the pre ⁇ ence of 2 mM ATP, 5 mM MgCl 2 in 50 mM Tri ⁇ -HCl (pH 7.6) . After precipitation with ammonium sulfate to 38% saturation, the pellet was collected at 10,000 x g for 10 minutes, suspended in Buffer A, and isolated by Mono-Q anion exchange and ⁇ uperose 6 chromatography.
  • the 0-38% precipitated material was then separated using Mono-Q anion exchange and each fraction as ⁇ ayed for its ability to influence the proteasome activity against Suc-LLVY-MCA or 125 I-lysozyme.
  • Column fractions were preincubated with the proteasome for 10 minutes and then either substrate was added. None of the column fractions by it ⁇ elf showed significant hydrolytic activity.
  • a peak of inhibitory activity was eluted around 240 to 280 mM NaCl. It significantly decreased proteolytic activity against both substrate ⁇ . Hydrolysis of lysozyme and the peptide was inhibited to a similar extent.
  • the active fraction ⁇ were pooled and chromatographed by gel filtration. The inhibitor eluted as a sharp peak with an apparent molecular weight of about 100-150 kDa.
  • proteasome activity decreased in a linear manner with both 125 I-lysozyme and Suc-LLVY- MCA as substrates, although the degree of the inhibition was highly variable between preparations.
  • the Inhibitor I ⁇ A Component of the l,500kDa Proteolytic Complex Like the inhibitor, one component of the 1,500 kDa proteolytic complex (CF-2) has been reported to have a molecular weight of about 250 kDa.
  • the inhibitor corresponds to CF-2
  • the inhibitor obtained by gel filtration was subjected to phosphocellulose chromatography.
  • Eytan et al . had noted that CF-2 has little affinity for phosphocellulose and elutes with le ⁇ s than 100 mM NaCl. Accordingly, Applicants found that the inhibitory activity wa ⁇ recovered in the flow through and 20 mM NaCl eluate (i.e., in the region where CF-2 activity was reported) .
  • CF-2 One unu ⁇ ual property of CF-2 is that it i ⁇ quite labile upon heating to 42°C, but i ⁇ ⁇ tabilized by ATP (Ganoth et al . , J. Biol . Chem . 263:12412-12419 (1988)) .
  • the inhibitor of the protea ⁇ ome corresponds to CF-2
  • the purified inhibitor was preincubated at 42 *C with or without ATP or the nonhydrolyzable analog, AMPPNP.
  • the protea ⁇ ome was added and after 10 minutes, peptidase activity wa ⁇ assayed. The degree of inhibition decreased rapidly during preincubation without nucleotide added. The pre ⁇ ence of either ATP or AMPPNP prevented this loss of activity.
  • ATP stabilizes the inhibitory factor, it is not essential for inhibition of the proteasome. After preincubation of the inhibitor with proteasome for up to 20 minutes with or without ATP, a similar degree of inhibition was observed. Nevertheless, because of the stabilization by ATP, this nucleotide was routinely added to all incubations.
  • the inhibitor preparations When analyzed by SDS-PAGE, the inhibitor preparations showed a major band of 40 kDa. To test whether this 40 kDa subunit corresponded to any subunit of the 1,500 kDa complex, the 1,500 kDa complex was formed by incubation of fraction II with Mg -ATP and isolated by anion exchange and gel filtration chromatography. SDS-PAGE of these active fraction ⁇ indicated many polypeptide ⁇ ⁇ imilar to those previously reported for thi ⁇ complex (Hough et al . , J. Biol . Chem . 262:8303-8313 (1987) ; Ganoth et al . , J . Biol . Chem .
  • fraction II was immunoprecipitated using an anti-proteasome monoclonal antibody and analyzed by SDS-PAGE. Ub-conjugate degrading activity had previously been ⁇ hown to be removed upon immunoprecipitation of fraction II (Matthews et al . , Proc . Na tl . Adac . Sci .
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox

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Abstract

L'invention concerne des procédés et des principes actifs qui inhibent le processus de présentation d'antigènes par des molécules de classe I du système majeur d'histocompabilité. Plus particulièrement, les inhibiteurs de la voie protéolytique dépendant de l'ubiquitine ATP pouvant inhiber la présentation de l'antigène CHM-1 sont décrits. Ces procédés et principes actifs peuvent être utilisés pour le traitement de maladies auto-immunes et pour limiter le rejet d'organes et d'implants.
PCT/US1994/001183 1993-02-10 1994-01-27 Role de la proteolyse dependant de l'ubiquitine atp dans la presentation de l'antigene restreint chm-1 et inhibiteurs utilises a cet effet WO1994017816A1 (fr)

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AU61691/94A AU676721B2 (en) 1993-02-10 1994-01-27 Role of ATP-ubiquitin-dependent proteolysis in MHC-1 restricted antigen presentation and inhibitors thereof
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WO1999022729A1 (fr) * 1997-10-31 1999-05-14 Centre De Recherche Du Centre Hospitalier De L'universite De Montreal Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique
WO1999031252A1 (fr) * 1997-12-12 1999-06-24 Incyte Pharmaceuticals, Inc. Proteine de conjugaison du type ubiquitine
FR2779653A1 (fr) * 1998-06-11 1999-12-17 Inst Nat Sante Rech Med Utilisation de composes modulateurs du proteasome en therapie
US6287569B1 (en) 1997-04-10 2001-09-11 The Regents Of The University Of California Vaccines with enhanced intracellular processing
US6613541B1 (en) 1998-10-20 2003-09-02 Millennium Pharmaceuticals, Inc. Method for monitoring proteasome inhibitor drug action
US7223745B2 (en) 2003-08-14 2007-05-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
EP1997823A1 (fr) 1994-10-28 2008-12-03 Millennium Pharmaceuticals, Inc. Composés d'acide et ester boronique, synthèse et utilisations
US7576206B2 (en) 2003-08-14 2009-08-18 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8168803B2 (en) 2003-06-20 2012-05-01 Nereus Pharmaceuticals, Inc. Methods of using [3.2.0] heterocyclic compounds and analogs thereof
US8283367B2 (en) 2005-02-11 2012-10-09 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8541590B2 (en) 2009-12-22 2013-09-24 Cephalon, Inc. Proteasome inhibitors and processes for their preparation, purification and use

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JP2002105080A (ja) * 2000-09-29 2002-04-10 Microbial Chem Res Found 生理活性物質パネポフェナンスリンとその製造法

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Am. J. Respir. Cell Mol. Biol., Vol. 7, issued 1992, A.L. SCHWARTZ et al., "Ubiquitin-Mediated Protein Modification and Degradation", pages 463-468, see Abstract. *
Ann. Rev. Cell Biol., Vol. 3, issued 1987, M. RECHSTEINER, "Ubiquitin-Mediated Pathways for Intracellular Proteolysis", pages 1-30, see entire document. *
Biochem. Soc. Trans., Vol. 19, No. 3, issued 1991, A. HERSHKO, "The Ubiquitin Pathway of Protein Degradation and Proteolysis of Ubiquitin-Protein Conjugates", pages 726-9, see Abstract. *
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Proc. Natl. Acad. Sci. (USA), Vol. 89, issued September 1992, L.D. FALO et al., "Serum Proteases Alter the Antigenicity of Peptides Presented by Class I Major Histocompatibility Complex Molecules", pages 8347-8350, see Abstract. *
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EP1997823A1 (fr) 1994-10-28 2008-12-03 Millennium Pharmaceuticals, Inc. Composés d'acide et ester boronique, synthèse et utilisations
US6287569B1 (en) 1997-04-10 2001-09-11 The Regents Of The University Of California Vaccines with enhanced intracellular processing
WO1999022729A1 (fr) * 1997-10-31 1999-05-14 Centre De Recherche Du Centre Hospitalier De L'universite De Montreal Utilisation d'inhibiteurs de proteasomes dans le traitement du cancer, de l'inflammation, de maladies autoimmunes, du rejet du greffon et du choc septique
WO1999031252A1 (fr) * 1997-12-12 1999-06-24 Incyte Pharmaceuticals, Inc. Proteine de conjugaison du type ubiquitine
FR2779653A1 (fr) * 1998-06-11 1999-12-17 Inst Nat Sante Rech Med Utilisation de composes modulateurs du proteasome en therapie
US6613541B1 (en) 1998-10-20 2003-09-02 Millennium Pharmaceuticals, Inc. Method for monitoring proteasome inhibitor drug action
US8168803B2 (en) 2003-06-20 2012-05-01 Nereus Pharmaceuticals, Inc. Methods of using [3.2.0] heterocyclic compounds and analogs thereof
US7915236B2 (en) 2003-08-14 2011-03-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7576206B2 (en) 2003-08-14 2009-08-18 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8058262B2 (en) 2003-08-14 2011-11-15 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US7223745B2 (en) 2003-08-14 2007-05-29 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8546608B2 (en) 2003-08-14 2013-10-01 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US9233115B2 (en) 2003-08-14 2016-01-12 Millennium Pharmaceuticals Inc. Proteasome inhibitors and methods of using the same
US8283367B2 (en) 2005-02-11 2012-10-09 Cephalon, Inc. Proteasome inhibitors and methods of using the same
US8541590B2 (en) 2009-12-22 2013-09-24 Cephalon, Inc. Proteasome inhibitors and processes for their preparation, purification and use

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