Adenovirus L4-100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death.

Cytotoxic lymphocytes kill virus-infected target cells and play a critical role in host recovery from viral infections. Granzyme B (GrB) is a cytotoxic lymphocyte granule protease that plays a critical role in mediating cytotoxicity. In these studies, we demonstrate that the adenovirus assembly protein L4--100K (100K) is a GrB substrate that prevents cytotoxic lymphocyte granule-induced apoptosis in infected target cells by potently inhibiting GrB. This inhibition is absolutely dependent on Asp-48 in 100K, found within a classic GrB consensus motif. 100K is the first viral protein described that exclusively targets the GrB pathway. It represents a novel class of viral protease inhibitor, in which an essential, multifunctional viral protein, which is vulnerable to specific proteolysis by GrB, expresses inhibitory function against that protease.

The DNA mismatch repair enzyme PMS1 is a myositis-specific autoantigen.

OBJECTIVE: The specificity of the autoantibody response in different autoimmune diseases makes autoantibodies useful for diagnostic purposes. It also focuses attention on tissue- and event-specific circumstances that may select unique molecules for an autoimmune response in specific diseases. Defining additional phenotype-specific autoantibodies may identify such circumstances. This study was undertaken to investigate the disease specificity of PMS1, an autoantigen previously identified in some sera from patients with myositis. METHODS: We used immunoprecipitation analysis to determine the frequency of autoantibodies to PMS1 in sera from patients with myositis, systemic lupus erythematosus, or scleroderma and from healthy controls. Additional antigens recognized by PMS1-positive sera were further characterized in terms of their susceptibility to cleavage by apoptotic proteases. RESULTS: PMS1, a DNA mismatch repair enzyme, was identified as a myositis-specific autoantigen. Autoantibodies to PMS1 were found in 4 of 53 patients with autoimmune myositis (7.5%), but in no sera from 94 patients with other systemic autoimmune diseases (P = 0.016). Additional mismatch repair enzymes (PMS2, MLH1) were targeted, apparently independently. Sera recognizing PMS1 also recognized several other proteins involved in DNA repair and remodeling, including poly(ADP-ribose) polymerase, DNA-dependent protein kinase, and Mi-2. All of these autoantigens were efficiently cleaved by granzyme B, generating unique fragments not observed during other forms of cell death. CONCLUSION: PMS1 autoantibodies are myositis specific. The striking correlation between an immune response to a group of granzyme B substrates (functioning in DNA repair and remodeling) and the myositis phenotype strongly implies that tissue- and event-specific biochemical events play a role in selecting these molecules for an autoimmune response. Understanding the role of granzyme B cleavage in this response is an important priority.

Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis.

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.

The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling.

Caspase-3-mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis.

Proteolytic cleavage of key substrates appears to be an important biochemical mechanism underlying the apoptotic process, and the centrality of interleukin 1 beta-converting enzyme (ICE)-like proteases as mediators of apoptosis has been suggested. The identification of the relevant substrates of the ICE protease family during apoptosis therefore constitutes a major challenge. Using human autoantibodies, we demonstrate here that a subset of autoantigens is specifically cleaved early during apoptosis. One of these cleaved molecules is identified as the catalytic subunit of the DNA-dependent protein kinase. The time courses of all proteolytic cleavages are identical and coincide with the onset of morphologic apoptosis. Furthermore, all cleavages share the same inhibition characteristics, which implicate an ICE-like activity(ies). We propose that cleavage of these autoantigens targets these molecules for an autoimmune response by revealing immunocryptic fragments in a proimmune apoptotic setting. Study of the immunogenicity of these fragments may yield insights into the autoimmune targeting of molecules. Moreover, the autoantibodies described will be valuable tools for the elucidation of mechanistically important proteolytic steps along the apoptotic pathway.

Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death.

The U1 small nuclear ribonucleoprotein particle is essential for splicing of precursor mRNA, an activity that depends upon both the RNA and protein components of the U1 particle. One of the U1-specific proteins that is functionally important in this splicing reaction is the 70-kDa protein (U1-70kDa). We report here that U1-70kDa is specifically cleaved in apoptotic cells, resulting in the generation of a 40-kDa fragment. The kinetics of this cleavage coincided with the appearance of cells with apoptotic morphology in the population, and the proportion of 40-kDa fragment observed was markedly increased in apoptotic cells that had become detached from the substratum. Although the inhibitor characteristics of the activity cleaving U1-70kDa suggest that interleukin 1 beta-converting enzyme (ICE) might be responsible, the specific ICE inhibitor N-(N-acetyl-tyrosinyl-valinyl-alaninyl)-3-amino-4-oxob utanoic acid (YVAD-CHO) did not prevent cleavage, and U1-70kDa was not cleaved by purified ICE in vitro. Further study of this novel cleavage and the enzyme responsible will yield information about proteolytic events that might be central in the mechanism and control of apoptosis.

Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.

Systemic lupus erythematosus is a multisystem autoimmune disease in which the autoantibody response targets a variety of autoantigens of diverse subcellular location. We show here that these autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented endoplasmic reticulum (ER) and ribosomes, as well as the ribonucleoprotein, Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. These autoantigen clusters have in common their proximity to the ER and nuclear membranes, sites of increased generation of reactive oxygen species in apoptotic cells. Oxidative modification at these sites may be a mechanism that unites this diverse group of molecules together as autoantigens.

Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.

We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.

Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase.

We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.

Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery.

We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.