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1.
EMBO J ; 26(5): 1385-96, 2007 Mar 07.
Article in English | MEDLINE | ID: mdl-17318184

ABSTRACT

Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10-30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases.


Subject(s)
Aminopeptidases/metabolism , Peptide Hydrolases/metabolism , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Aminopeptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Endopeptidases/genetics , Endopeptidases/metabolism , HeLa Cells , Humans , Leucyl Aminopeptidase/metabolism , Models, Biological , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptides/chemistry , RNA, Small Interfering/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
Biochem Soc Trans ; 35(Pt 1): 12-7, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17212580

ABSTRACT

This review focuses on recent insights into the mechanisms and the biological functions of the proteasome. This large ATP-dependent proteolytic complex is the main site for protein degradation in mammalian cells and catalyses the rapid degradation of ubiquitinated proteins, and is the source of most antigenic peptides used by the immune system to screen for viruses and cancer. ATP is required to unfold globular proteins to open the gated channel into the 20S proteasome and to facilitate protein translation into it. Inhibitors of its proteolytic activity are widely used as research tools and have proven effective in cancer therapy.


Subject(s)
Antineoplastic Agents/therapeutic use , Immune System/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Proteasome Endopeptidase Complex/physiology , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , Catalysis , Humans , Models, Biological , Models, Chemical , Proteasome Endopeptidase Complex/chemistry , Ubiquitin/chemistry
3.
Proc Natl Acad Sci U S A ; 98(25): 14440-5, 2001 Dec 04.
Article in English | MEDLINE | ID: mdl-11717410

ABSTRACT

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.


Subject(s)
Ligases/genetics , Muscle Proteins/genetics , Muscular Atrophy/genetics , SKP Cullin F-Box Protein Ligases , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Fasting/metabolism , Gene Expression , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism
4.
Chem Biol ; 8(8): 739-58, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11514224

ABSTRACT

The 26S proteasome is a 2.4 MDa multifunctional ATP-dependent proteolytic complex, which degrades the majority of cellular polypeptides by an unusual enzyme mechanism. Several groups of proteasome inhibitors have been developed and are now widely used as research tools to study the role of the ubiquitin-proteasome pathway in various cellular processes, and two inhibitors are now in clinical trials for treatment of multiple cancers and stroke.


Subject(s)
Peptide Hydrolases/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Proteasome Endopeptidase Complex , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Anti-HIV Agents/therapeutic use , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Binding Sites , Catalysis , Humans , Models, Molecular , Multienzyme Complexes/metabolism , Protease Inhibitors/classification , Protease Inhibitors/therapeutic use , Protein Structure, Quaternary , Protein Subunits , Ubiquitin/metabolism
5.
Curr Opin Clin Nutr Metab Care ; 4(3): 183-90, 2001 May.
Article in English | MEDLINE | ID: mdl-11517350

ABSTRACT

Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.


Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Muscular Atrophy/etiology , Ubiquitins/metabolism , Animals , Culture Techniques , Cysteine Endopeptidases/genetics , Cysteine Proteinase Inhibitors/pharmacology , Humans , Multienzyme Complexes/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Muscular Atrophy/enzymology , Muscular Atrophy/metabolism , Proteasome Endopeptidase Complex , RNA, Messenger/metabolism , Ubiquitins/genetics , Wasting Syndrome/etiology , Wasting Syndrome/metabolism
6.
J Biol Chem ; 276(39): 36474-81, 2001 Sep 28.
Article in English | MEDLINE | ID: mdl-11479311

ABSTRACT

Nearly all peptides generated by proteasomes during protein degradation are digested rapidly to amino acids, but a few proteasomal products escape this fate and are presented to the immune system on cell surface major histocompatibility complex class I molecules. To test whether these antigenic peptides may be inherently resistant to cytosolic peptidases, six different antigenic peptides were incubated with HeLa cell extracts. All six were degraded rapidly by a process involving o-phenanthroline-sensitive metallopeptidases. One antigenic peptide, FAPGNYPAL, was rapidly destroyed in the extracts by a bestatin-sensitive exopeptidase, apparently by the puromycin-sensitive aminopeptidase. The disappearance of the other five was reduced 30-90% by a specific inhibitor of the cytosolic endopeptidase, thimet oligopeptidase (TOP) (EC ), whose physiological function(s) have been unclear and controversial. All these peptides were sensitive to pure recombinant TOP. Furthermore, upon fractionation of the extracts, the major peptidase peak that degraded the ovalbumin-derived epitope, SIINFEKL, co-purified with TOP. In the extracts, TOP also catalyzed rapid degradation of N-extended variants of SIINFEKL and of other antigenic peptides, which in vivo can serve as precursors of these major histocompatibility complex-presented epitopes. This enzyme (unlike cell proteins that promote production of antigenic peptides) is not regulated by interferon-gamma. TOP seems to be primarily responsible for the rapid breakdown of antigenic peptides in cytosolic extracts, and our related studies (A. X. Y. Mo, K. Lemerise, W. Zeng, Y. Shen, C. R. Abraham, A. L. Goldberg, and K. L. Rock, submitted for publication) indicate that TOP by destroying such peptides limits antigen presentation in vivo.


Subject(s)
Antigens/chemistry , Genes, MHC Class I , Major Histocompatibility Complex , Metalloendopeptidases/chemistry , Peptides/chemistry , Amino Acids/chemistry , Antigen Presentation , Catalysis , Chromatography, High Pressure Liquid , HeLa Cells , Humans , Immunoblotting , Interferon-gamma/chemistry , Leucine/analogs & derivatives , Leucine/pharmacology , Protease Inhibitors/pharmacology , Protein Structure, Tertiary , Protein Synthesis Inhibitors/pharmacology , Puromycin/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Time Factors
7.
Mol Cell ; 7(6): 1143-52, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11430818

ABSTRACT

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.


Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cysteine Endopeptidases/metabolism , Ion Channel Gating/physiology , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Antigen Presentation/physiology , Cysteine Endopeptidases/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Ion Channel Gating/drug effects , Molecular Sequence Data , Multienzyme Complexes/chemistry , Peptide Hydrolases/chemistry , Potassium Channels/pharmacology , Proteasome Endopeptidase Complex , Protein Structure, Quaternary , Substrate Specificity , Yeasts
8.
EMBO J ; 20(10): 2357-66, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11350924

ABSTRACT

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).


Subject(s)
Antigens/biosynthesis , Egg Proteins/biosynthesis , Epitopes, B-Lymphocyte/biosynthesis , Immunodominant Epitopes/biosynthesis , Ovalbumin/biosynthesis , Peptide Biosynthesis , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Animals , Antigens/immunology , Egg Proteins/immunology , Epitopes, B-Lymphocyte/immunology , Hybridomas , Immunodominant Epitopes/immunology , Mice , Ovalbumin/immunology , Peptide Fragments , Peptides/immunology
9.
J Biol Chem ; 276(26): 24261-7, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11301331

ABSTRACT

The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins. Here we report a new role for trehalose in protecting cells against oxygen radicals. Exposure of Saccharomyces cerevisiae to a mild heat shock (38 degrees C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H(2)O(2)/iron). When cells were returned to normal growth temperature (28 degrees C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly. Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells. Providing trehalose exogenously enhanced the resistance of mutant cells to H(2)O(2). Exposure of cells to H(2)O(2) caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage. After even brief exposure to H(2)O(2), the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells. Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger. Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.


Subject(s)
Oxidative Stress , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/metabolism , Trehalose/biosynthesis , Trehalose/physiology , Cell Survival/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Heat-Shock Response , Hydrogen Peroxide/pharmacology , Leupeptins/pharmacology , Mutation , Oxidation-Reduction , Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Trehalose/genetics
10.
Biochimie ; 83(3-4): 311-8, 2001.
Article in English | MEDLINE | ID: mdl-11295491

ABSTRACT

26S proteasomes are composed of a 20S proteolytic core and two ATPase-containing 19S regulatory particles. To clarify the role of these ATPases in proteolysis, we studied the PAN complex, the archaeal homolog of the 19S ATPases. When ATP is present, PAN stimulates protein degradation by archaeal 20S proteasomes. PAN is a molecular chaperone that catalyzes the ATP-dependent unfolding of globular proteins. If 20S proteasomes are present, this unfoldase activity is linked to degradation. Thus PAN, and presumably the 26S ATPases, unfold substrates and facilitate their entry into the 20S particle. 26S proteasomes preferentially degrade ubiquitinated proteins. However, we found that calmodulin (CaM) and troponin C are degraded by 26S proteasomes without ubiquitination. Ca(2+)-free native CaM and in vitro 'aged' CaM are degraded faster than the Ca(2+)-bound form. Ubiquitination of CaM does not enhance its degradation. Degradation of ovalbumin normally requires ubiquitination, but can occur without ubiquitination if ovalbumin is denatured. The degradation of these proteins still requires ATP and the 19S particle. Thus, ubiquitin-independent degradation by 26S proteasomes may be more important than generally assumed.


Subject(s)
Adenosine Triphosphatases/metabolism , Cysteine Endopeptidases/metabolism , Molecular Chaperones/metabolism , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Animals , Calcium/chemistry , Calcium/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , Endopeptidases/metabolism , Humans , Ovalbumin/chemistry , Ovalbumin/metabolism , Proteasome Endopeptidase Complex , Protein Denaturation/physiology , Protein Folding , Ubiquitins/metabolism
13.
J Biol Chem ; 276(6): 3920-8, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11062236

ABSTRACT

In addition to promoting protein folding and translocation, molecular chaperones of Hsp70/DnaJ families are essential for the selective breakdown of many unfolded proteins. It has been proposed that chaperones function in degradation to maintain the substrates in a soluble form. In Escherichia coli, a nonsecreted alkaline phosphatase mutant that lacks its signal sequence (PhoADelta2-22) fails to fold in the cytosol and is rapidly degraded at 37 degrees C. We show that PhoADelta2-22 is degraded by two ATP-dependent proteases, La (Lon) and ClpAP, and breakdown by both is blocked in a dnaJ259-ts mutant at 37 degrees C. Both proteases could be immunoprecipitated with PhoA, but to a much lesser extent in the dnaJ mutant. Therefore, DnaJ appears to promote formation of protease-substrate complexes. DnaJ could be coimmunoprecipitated with PhoA, and the extent of this association directly correlated with its rate of degradation. Although PhoA was not degraded when DnaJ was inactivated, 50% or more of the PhoA remained soluble. PhoA breakdown and solubility did not require ClpB. PhoA degradation was reduced in a thioredoxin-reductase mutant (trxB), which allowed PhoADelta2-22 to fold into an active form in the cytosol. Introduction of the dnaJ mutation into trxB cells further stabilized PhoA, increased enzyme activity, and left PhoA completely soluble. Thus, DnaJ, although not necessary for folding (or preventing PhoA aggregation), is required for PhoA degradation and must play an active role in this process beyond maintaining the substrate in a soluble form.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/metabolism , Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Adenosine Triphosphate/metabolism , Escherichia coli Proteins , HSP40 Heat-Shock Proteins , Hydrolysis , Protein Folding , Solubility
14.
Mol Cell ; 8(6): 1339-49, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11779508

ABSTRACT

The 19S component of the 26S proteasome contains six ATPase subunits. To clarify how they unfold and translocate proteins into the 20S proteasome for degradation, we studied the homologous archaebacterial proteasome-regulatory ATPase complex PAN and the globular substrate GFP-SsrA. When we attached a small (Biotin) or large (Biotin-Avidin) moiety near its N terminus or a Biotin near its C terminus, GFP-SsrA was unfolded and degraded. However, attaching Avidin near its C terminus blocked passage through PAN and prevented GFP-SsrA degradation. Though not translocated, GFP-Avidin still underwent ATP-dependent unfolding. Moreover, it remained bound to PAN and inhibited further proteolysis. Therefore, (1) translocation and degradation of this substrate require threading through the ATPase in a C to N direction and (2) translocation does not cause but follows ATP-dependent unfolding, which occurs on the surface of the ATPase ring.


Subject(s)
Adenosine Triphosphatases/metabolism , Archaea/enzymology , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Protein Folding , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/metabolism , Archaea/metabolism , Avidin/metabolism , Biotin/metabolism , Catalysis , Protein Denaturation , Protein Processing, Post-Translational , Protein Subunits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
15.
Nat Cell Biol ; 2(11): 833-9, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11056539

ABSTRACT

The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.


Subject(s)
Adenosine Triphosphatases/metabolism , Endopeptidases/metabolism , Methanococcus/enzymology , Molecular Chaperones/metabolism , Protein Folding , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Archaeal Proteins , Enzyme Activation , Magnesium , Molecular Chaperones/genetics , Protein Denaturation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Thermoplasma
16.
J Clin Invest ; 106(3): 439-48, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10930447

ABSTRACT

Induction of NF-kappaB-dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the alpha 7 subunit of the 26S proteasome and blocks degradation of NF-kappa B inhibitor I kappa B alpha by the ubiquitin-proteasome pathway without affecting overall proteasome activity. I kappa B alpha phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of I kappa B alpha degradation abolishes induction of NF-kappa B-dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-kappa B-dependent gene expression for therapeutic purposes.


Subject(s)
Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , I-kappa B Proteins , Multienzyme Complexes/metabolism , Peptides/pharmacology , Ubiquitins/antagonists & inhibitors , Animals , Anti-Infective Agents/metabolism , Cells, Cultured , Gene Expression/drug effects , Humans , Male , Mice , Mice, Inbred ICR , Mice, Transgenic , Myocardial Infarction/drug therapy , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Pancreatitis/drug therapy , Pancreatitis/genetics , Pancreatitis/metabolism , Peptides/genetics , Peptides/metabolism , Proteasome Endopeptidase Complex , Swine , Ubiquitins/metabolism
17.
Skeletal Radiol ; 29(3): 125-32, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10794549

ABSTRACT

OBJECTIVE: Computed tomography (CT) has been demonstrated to be superior to radiography in identifying cervical vertebral injuries. However, many of these injuries may not be clinically significant, and require only minimal symptomatic and supportive treatment. It is therefore imperative that radiologists and spine surgeons have criteria for distinguishing between those injuries requiring surgical stabilization and those that do not. The authors propose a new classification of cervical vertebral injuries into two categories: major and minor. DESIGN AND PATIENTS: A data base, acquired on 1052 separate cervical injuries in 879 patients seen between 1983 and 1998, was reviewed. Four categories of injury based on mechanism [hyperflexion (four variants), hyperextension (two variants), rotary (two variants), and axial compression (five variants)] were identified. "Major" injuries are defined as having either radiographic or CT evidence of instability with or without associated localized or central neurologic findings, or have the potential to produce the latter. "Minor" injuries have no radiographic and/or CT evidence of instability, are not associated with neurologic findings, and have no potential to cause the latter. RESULTS AND CONCLUSIONS: Cervical injury should be classified as "major" if the following radiographic and/or CT criteria are present: displacement of more than 2 mm in any plane, wide vertebral body in any plane, wide interspinous/interlaminar space, wide facet joints, disrupted posterior vertebral body line, wide disc space, vertebral burst, locked or perched facets (unilateral or bilateral), "hanged man" fracture of C2, dens fracture, and type III occipital condyle fracture. All other types of fractures may be considered "minor".


Subject(s)
Cervical Vertebrae/injuries , Spinal Injuries/classification , Spinal Injuries/diagnostic imaging , Humans , Spinal Injuries/etiology , Tomography, X-Ray Computed
18.
J Biol Chem ; 275(27): 20295-301, 2000 Jul 07.
Article in English | MEDLINE | ID: mdl-10791958

ABSTRACT

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.


Subject(s)
Calmodulin/metabolism , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex , Protein Denaturation , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Binding Sites , Calcium/metabolism , Calcium/pharmacology , Chickens , HeLa Cells , Humans , Microinjections , Muscle, Skeletal/enzymology , Oocytes/metabolism , Protein Conformation , Rabbits , Ubiquitins/metabolism , Xenopus
20.
J Biol Chem ; 275(20): 14831-7, 2000 May 19.
Article in English | MEDLINE | ID: mdl-10809725

ABSTRACT

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.


Subject(s)
Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Serine , Threonine , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Kinetics , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptide Library , Proteasome Endopeptidase Complex , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , Thermoplasma/enzymology
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