Mutations in IMPDH1 account for approximately 2% of families with adRP, and de novo IMPDH1 mutations are also rare causes of isolated LCA. This analysis of the novel IMPDH1 mutants substantiates previous reports that IMPDH1 mutations do not alter enzyme activity and demonstrates that these mutants alter the recently identified single-stranded nucleic acid binding property of IMPDH. Studies are needed to further characterize the functional significance of IMPDH1 nucleic acid binding and its potential relationship to retinal degeneration.

MHCII proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The non-classical MHCII protein HLA-DM chaperones peptide-free MHCII against inactivation and catalyzes peptide exchange on loaded MHCII. Another non-classical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we use x-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the alpha subunit 310 helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis demonstrates that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic and place constraints on possible functional roles for HLA-DO in antigen presentation.

Two mutations of IMPDH1 (inosine 5'-monophosphate dehydrogenase type I), R224P and D226N, have recently been found to cause adRP (autosomal dominant retinitis pigmentosa). IMPDH1 catalyses the rate-limiting step in guanine nucleotide biosynthesis and also binds single-stranded nucleic acids. In the present paper, we report the biochemical characterization of the adRP-linked mutations, R224P and D226N, and a potentially pathogenic mutation, V268I. The adRP-linked mutations have no effect on enzyme activity, protein stability or protein aggregation. These results suggest strongly that the mutations do not affect enzyme activity in vivo and thus do not perturb the guanine nucleotide pool. The R224P mutation changes the distribution of enzyme between the nucleus and cytoplasm. This effect was not observed with the D226N mutation, so the relevance of this observation to disease is unclear. In contrast, both mutations decrease the affinity of nucleic acid binding and both fail to co-immunoprecipitate RNA. These observations suggest that nucleic acid binding provides a functional assay for adRP pathogenicity. The putative adRP-linked mutation V268I also disrupts nucleic acid binding, which suggests that this mutation is indeed pathogenic.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.

IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation. IMP dehydrogenase (IMPDH)2 catalyzes the reaction that controls the entry of purines into the guanine nucleotide pool, and thus controls proliferation (1). The enzyme is a homotetramer; each monomer is composed of a catalytic (␤/␣) 8 barrel and a subdomain containing two CBS domains (named for the related domain in cystathionine ␤-synthase) (Fig. 1). Deletion of the subdomain has no effect on enzymatic activity (2, 3), and the function of the subdomain in IMPDH is currently under debate. CBS domains act as adenosine nucleotide-binding modules in several proteins (4 -9), and a similar role has been proposed for the CBS domains of IMPDH (5), but we and others have been unable to confirm this function in IMPDH (10 -13). Notably, the CBS domains of IMPDH share little sequence identity with the other proteins, so it would not be surprising if their function has diverged. The subdomain does appear to coordinately regulate the adenine and guanine nucleotide pool in Escherichia coli, although the molecular mechanism of this process has not yet been elucidated (11). We have discovered that IMPDH binds single-stranded nucleic acids and that the subdomain mediates this interaction (10, 15). IMPDH associates with RNA in tissue culture cells, which suggests that this housekeeping enzyme is involved in translation, splicing, or some other feature of RNA metabolism (10, 15). Others report that IMPDH binds DNA and may be involved in gene expression (16, 17). These observations suggest that IMPDH has a "moonlighting" function involving nucleic acid that is mediated by the subdomain.Mammals have two IMPDH genes, encoding IMPDH1 and IMPDH2, and most tissues express both isozymes (18,19). In contrast, only IMPDH1 appears to be expressed in the retina; in addition, retina contains distinct IMPDH1 isoforms generated by alternative mRNA splicing as follows: IMPDH1(546) (major) and IMPDH1(595) (minor) ( Fig. 1; these proteins are also known as IMPDH1␣/IMPDH1(13b) and IMPDH␥/IMPDH1(Aϩ13b), respectively; the canonical enzyme is hereaft...

HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/ sDM complex and have implications for the mechanism of DO inhibition of DM.antigen processing | antigen presentation | MHC class II M ajor histocompatibility complex (MHC) class II molecules present peptides on the antigen-presenting cell (APC) surface to sensitize CD4 + lymphocytes. The class II presentation pathway is well-characterized and includes roles for three accessory molecules: invariant chain (Ii), HLA-DM (DM), and HLA-DO (DO) (1). Newly synthesized MHC class II molecules bind to Ii in the ER. A trafficking signal in the cytoplasmic domain of invariant chain directs the complex to late endosomal compartments (2, 3), called MIIC (MHC II containing compartments). The invariant chain (Ii) is proteolytically degraded in this low-pH environment, leaving a nested set of invariant chain fragments, CLIPs (class II-associated invariant chain peptides), in the class II peptide-binding groove (4). CLIP is ultimately exchanged for antigenic peptides by DM, a nonclassic MHC class II molecule, which further influences peptide selection in a process termed "peptide editing" (5-10). DM also stabilizes empty MHC class II to maintain a peptide receptive structure; otherwise, empty MHC class II proteins become peptide averse (11).DO, another nonclassic MHC class II αβ heterodimer, first was described as an inhibitor of DM function, because overexpression of DO in human cells increased levels of CLIP-bound class II molecules (12, 13). In vitro evidence most often corroborated the view of DO as a negative modulator of DM (12,14). Results suggesting that DO inhibition of DM is robust at early endosomal pH (6.0-6.5) and attenuated at the lower pH (4.5-5.0) of late endosomal/lysosomal MHC II containing compartments...

The objective of this study is to determine how a hibernating mammal avoids the formation of blood clots under periods of low blood flow. A microfluidic vascular injury model was performed to differentiate the effects of temperature and shear rate on platelet adhesion to collagen. Human and ground squirrel whole blood was incubated at 15 or 37°C and then passed through a microfluidic chamber over a 250 μm strip of type I fibrillar collagen at that temperature and shear rates of 50 s−1 or 300 s−1 to simulate torpid and aroused conditions respectively. At 15°C, both human and ground squirrel platelets showed a 90–95% decrease in accumulation on collagen independent of shear rate. At 37°C, human platelet accumulation reduced by 50% at 50 s−1 compared to 300 s−1, while ground squirrel platelet accumulation dropped by 80%. When compared to platelets from non-hibernating animals, platelets from animals collected after arousal from torpor showed a 60% decrease in binding at 37°C and 300 s−1, but a 2.5-fold increase in binding at 15°C and 50 s−1. vWF binding in platelets from hibernating ground squirrels were decreased by 50% relative to non-hibernating platelets. The source of the plasma that platelets were stored in did not affect the results indicating that the decreased vWF binding was a property of the platelets. Upon chilling, ground squirrel platelets increase microtubule assembly leading to the formation of long rods. This shape change is concurrent with sequestration of platelets in the liver and not the spleen. In conclusion, it appears that ground squirrel platelets are sequestered in the liver during torpor, have reduced binding capacity for plasma vWF, and lower accumulation on collagen at low shear rates and after storage at cold temperatures, while still being activated by external agonists. These adaptations would protect the animals from spontaneous thrombus formation during torpor but allow them to restore normal platelet function upon arousal.

Titin contributes to sarcomere assembly, muscle signaling, and mechanical properties of muscle. The mdm mouse exhibits a small deletion in the titin gene resulting in dystrophic mutants and phenotypically normal heterozygotes. We examined the effects of this mutation on locomotion to assess how, and if, changes to muscle phenotype explain observed locomotor differences. Mutant mice are much smaller in size than their siblings and gait abnormalities may be driven by differences in limb proportions and/or by changes to muscle phenotype caused by the titin mutation. We quantified differences in walking gait among mdm genotypes and also determined whether genotypes vary in limb morphometrics. Mice were filmed walking, and kinematic and morphological variables were measured. Mutant mice had a smaller range of motion at the ankle, shorter stride lengths, and shorter stance duration, but walked at the same relative speeds as the other genotypes. Although phenotypically similar to wildtype mice, heterozygous mice frequently exhibited intermediate gait mechanics. Morphological differences among genotypes in hindlimb proportions were small and do not explain the locomotor differences. We suggest that differences in locomotion among mdm genotypes are due to changes in muscle phenotype caused by the titin mutation.