Evan Hermel

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78.

Alterations in Ca2+ homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose‐regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress‐induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c‐mediated caspase activation in a cell‐free system, and expression of GRP78 blocks both caspase activation and caspase‐mediated cell death. GRP78 forms a complex with caspase‐7 and ‐12 and prevents release of caspase‐12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase‐12 into the cytoplasm to set in motion the cytosolic component of the ER stress‐induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress‐induced cell death.

Maternally transmitted histocompatibility antigen of mice: A hydrophobic peptide of a mitochondrially encoded protein

MTF, a murine minor histocompatibility antigen, is maternally inherited and thought to be encoded by a mitochondrial gene. We sequenced the entire mitochondrial genomes from three strains that differ in MTF Mtf beta, Mtf gamma, and Mtf delta) and compared the sequences with the known, Mtf alpha, mitochondrial DNA sequence. We found only one site where all four genomes differed, affecting amino acid residue 6 of ND1, a subunit of NADH dehydrogenase. Incubation of non-Mtf alpha target cells with synthetic peptide ND1 alpha 1-17 (the first 17 amino acid of the ND1 protein of Mtf alpha mice) rendered them susceptible to lysis by MTF alpha-specific cytotoxic T cells (CTLs). Similarly, non-Mtf beta target cells were lysed by MTF beta-specific CTLs after incubation with the allelic form ND1 beta 1-17. Thus, Mtf is attributable to allelic variation at a single residue of the ND1 protein. Cells can therefore display peptides derived from mitochondrially encoded proteins, and such peptides can be histocompatibility antigens.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

AbstractHuntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Proteolytic Cleavage of Ataxin-7 by Caspase-7 Modulates Cellular Toxicity and Transcriptional Dysregulation

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.

Antigen Presentation by Neoclassical MHC Class I Gene Products in Murine Rodents

A number of cytotoxic T cell responses in rats and mice are restricted by medial or non- classical class I antigens of the major histocompatibility complex (MHC). The class I heavy chain encoded by the H-2M3 gene presents MTF, an N-formylated peptide derived from the amino terminus of the mitochondrially-encoded ND1 protein, which is polymorphic in the sixth residue. H-2M3 shows minimal polymorphism in mice, and it is more similar to its rat ortholog, RT1.M3, than to any other H-2 class I gene. In rats, a minor antigen is presented by an RT1.C-encoded restriction element. Other examples of antigen presentation by medial class I antigens are reviewed. Whereas the majority of medial class I genes have ill-defined, if any, function, we propose that some may serve to present special antigens, and that these neo-classical class I genes are highly conserved between species and may be localized at the distal end of the MHC.

Design, Synthesis and Evaluation of Indole Compounds as Novel Inhibitors targeting Gp41

A series of indole ring containing compounds were designed based on the structure of the gp41 complex in the region of the hydrophobic pocket. These compounds were synthesized using a Suzuki Coupling reaction, and evaluated using a fluorescence binding assay and cell-cell fusion assay. The observed inhibition constant of compound 7 was 2.1microM, and the IC(50) for cell-cell fusion inhibition was 1.1microM. Assay data indicated that 7 is a promising lead compound for optimization into an effective low molecular weight fusion inhibitor.

Sequence divergence of B2m alleles of wild Mus musculus and Mus spretus implies positive selection.

Mouse β2-microglobulin (β2m) is polymorphic. Sequences of five allelic wild mouse B2m genes have been determined from the large exons of genomic DNA using the polymerase chain reaction. Relative to the standard B2ma allele, the products of four alleles of Mus musculus origin (w2, w3, w4, and w5), differ by only one or two amino acids. w5 has a single nucleotide change, Asp85 → Val, and is identical to the c allele. w2 differs at Arg81 → Thr and w4 at His34 → Gln, and they share the Asp85 → Val change with B2mc and B2mw5.w5 and c cells are lysed by S19.8, a monoclonal antibody specific for β2mb (Ala85), in a complement-mediated cytotoxicity assay, whereas w4 cells are not. Thus, distant changes appear to introduce subtle conformational effects on β2m structure. Five independent isolates of Mus spretus (w1) differ the most from B2ma, with 12 amino acid changes and only one silent substitution. Replacements predicted from the nucleotide sequence occur in loops of the molecule facing away from the class I heavy chain and not in regions where β2m associates with class 1 α3 domains. Concordantly, the w1 – 5 allelic forms of β2m associate well with H-2 heavy chains. The many amino acid changes in the spretus sequence and the paucity of silent substitutions suggest that B2m has been subject to positive selection.

Polymorphism and conservation of the genes encoding Qa1 molecules

To evaluate the polymorphism and conservation of the major histocompatibility complex class Ib molecule Qa1 in wild mouse populations, we determined the nucleotide sequence of exons 1–3 of Qa1 of eight mouse haplotypes derived from wild mice, including Mus musculus domesticus, M. m. castaneus, M. m. bactrianus, and M. spretus, as well as two t haplotypes. Our data identify eight new alleles of Qa1. Taken together with previously published data on Qa1 among the common laboratory inbred strains, and in agreement with cytotoxic T-lymphocyte, serological, and biochemical data, these results further confirm the existence of two families of Qa1 molecules, Qa1a-like and Qa1b-like, and illuminate the extreme conservation of the peptide-binding region of these molecules, even across species.

RT1.DMa and RT1.DMb: the rat homologues of H2-DMA and H2-DMb

Proper class II antigen presentation is dependent upon the products of the M class II loci, HLA-DMA and HLA-DMB in humans; H2-DMa and H2-DMb in mice (Denzin et al. 1994; Fling et al. 1994; Morris et al. 1994; Denzin and Cresswell, 1995; Sloan et al. 1995). The DM class II proteins are no more than 30% similar to classical class II antigens such as HLA-DR or H2-E, but are clearly orthologous, being some 70% identical between humans and mice (Cho et al. 1991; Kelly et al. 1991). We reasoned that given the genetic similarity of mice and rats, we could isolate the rat homologues for H2-DMa and -DMb by use of the polymerase chain reaction (PCR) with primers originally used for identifying alleles of H2-DMa and H2-DMb (Hermel et al. 1995). We use the DM nomenclature for the mouse loci to avoid confusion with the class Ib H2-M genes. Liver RNA from an outbred Rattus norvegicus was reverse-transcribed and amplified by PCR. Products of 700 base pairs in size were obtained, cloned, and sequenced. The nucleotide sequences of RT1.DMa and -DMb are shown in Figure 1. Exons 2 and 3 of RT1.DMa differed from the mouse H2-DMa by 9.5% at the nucleotide level and 12.6% at the protein level (Fig. 2A). For exons 2 and 3, RT1.DMb differed by 10.9% and 9.8% from H2DMbl and H2-DMb2, respectively (Fig. 2B). In both genes, exon 2 contained nearly twice as many changes as did exon 3. For predicted protein sequences, the overall difference between RT1.DM[~ and both H2-DM[31 and H2DM~2 is 10%. This difference rises to 20% for the membrane distal domains of RT1.DMJ3 and H2-DMJ31, while the difference from DM[32 is 18.9%. It is unclear whether RT1.DM[3 is more closely related to H2-DM~I or H2-DM~2, as the membrane distal domain of RT1.DM[~ A

A possible mechanism for maintenance of the deleterious allele of human CASPASE-12

In humans, a functional CASPASE-12 (CASP12) gene has been identified only in persons of African heritage and has been suggested to play a regulatory role in response to bacterial pathogens and in promoting and increased susceptibility to sepsis. The existence of a gene whose effect is deleterious, and which has been the subject of extensive negative selection in the rest of the human population, implies the simultaneous presence of some selective benefit for persons having CASP12. Given the importance of inflammatory immune responses in controlling the initial stages of infection, and the role that CASP12 plays in down-regulating inflammation, we hypothesize that pathogens which exploit the inflammatory response are restrained by an active CASP12 gene product. Several candidate pathogens are discussed.