Serine racemase deletion alters adolescent social behavior and whole-brain cFos activation.

Background: Neurodevelopmental disorders (NDDs) can cause debilitating impairments in social cognition and aberrant functional connectivity in large-scale brain networks, leading to social isolation and diminished everyday functioning. To facilitate the treatment of social impairments, animal models of NDDs that link N- methyl-D-aspartate receptor (NMDAR) hypofunction to social deficits in adulthood have been used. However, understanding the etiology of social impairments in NDDs requires investigating social changes during sensitive windows during development. Methods: We examine social behavior during adolescence using a translational mouse model of NMDAR hypofunction (SR-/-) caused by knocking out serine racemase (SR), the enzyme needed to make D-serine, a key NMDAR coagonist. Species-typical social interactions are maintained through brain-wide neural activation patterns; therefore, we employed whole-brain cFos activity mapping to examine network-level connectivity changes caused by SR deletion. Results: In adolescent SR-/- mice, we observed disinhibited social behavior toward a novel conspecific and rapid social habituation toward familiar social partners. SR-/- mice also spent more time in the open arm of the elevated plus maze which classically points to an anxiolytic behavioral phenotype. These behavioral findings point to a generalized reduction in anxiety-like behavior in both social and non-social contexts in SR-/- mice; importantly, these findings were not associated with diminished working memory. Inter-regional patterns of cFos activation revealed greater connectivity and network density in SR-/- mice compared to controls. Discussion: These results suggest that NMDAR hypofunction - a potential biomarker for NDDs - can lead to generalized behavioral disinhibition in adolescence, potentially arising from disrupted communication between and within salience and default mode networks.

Diversity, equity, and inclusion correlates of racial/ethnic harassment and discrimination in the U.S. military.

As one of the most racially/ethnically diverse workplaces in the United States, the Department of Defense (DoD) has been on the forefront in driving diversity initiatives. Yet, racial/ethnic harassment and discrimination (REHD) in the military persist and threaten mission readiness. Despite this, limited research exists identifying factors that influence REHD in the U.S. military that could be leveraged for prevention and intervention. In this study, we sought to identify how diversity, equity, and inclusion (DEI) factors in the workplace are associated with REHD in order to identify potential targets for prevention and policy efforts to improve racial/ethnic relations in the U.S. military. Using the 2017 Workplace and Equal Opportunity Survey of Active Duty Members, we found military, leadership, and unit DEI climate factors were the top predictors of REHD, though the relative importance of each predictor varied by racial/ethnic minority status. In particular, we found military and leadership attention to REHD to be the top predictors for Racial/Ethnic Minority active duty members whereas workplace hostility was the top predictor for non-Hispanic White active duty members. Implications for programs and policies surrounding REHD in the U.S. military are discussed.

D-serine availability modulates prefrontal cortex inhibitory interneuron development and circuit maturation.

The proper development and function of telencephalic GABAergic interneurons is critical for maintaining the excitation and inhibition (E/I) balance in cortical circuits. Glutamate contributes to cortical interneuron (CIN) development via N-methyl-D-aspartate receptors (NMDARs). NMDAR activation requires the binding of a co-agonist, either glycine or D-serine. D-serine (co-agonist at many mature forebrain synapses) is racemized by the neuronal enzyme serine racemase (SR) from L-serine. We utilized constitutive SR knockout (SR-/-) mice to investigate the effect of D-serine availability on the development of CINs and inhibitory synapses in the prelimbic cortex (PrL). We found that most immature Lhx6 + CINs expressed SR and the obligatory NMDAR subunit NR1. At embryonic day 15, SR-/- mice had an accumulation of GABA and increased mitotic proliferation in the ganglionic eminence and fewer Gad1 + (glutamic acid decarboxylase 67 kDa; GAD67) cells in the E18 neocortex. Lhx6 + cells develop into parvalbumin (PV+) and somatostatin (Sst+) CINs. In the PrL of postnatal day (PND) 16 SR-/- mice, there was a significant decrease in GAD67+ and PV+, but not SST + CIN density, which was associated with reduced inhibitory postsynaptic potentials in layer 2/3 pyramidal neurons. These results demonstrate that D-serine availability is essential for prenatal CIN development and postnatal cortical circuit maturation.

Microaggressions: Mega problems or micro issues? A meta-analysis.

While research on microaggressions has accumulated in recent decades, doubts have arisen over their impact on individuals. Hence, the purpose of this study was to analyze the relations between microaggressions and psychological well-being, physical health, job outcomes, and positive and negative coping. Potential moderators (i.e., microaggression target, publication year, publication status, sample occupation, and inclusion of nonstigmatized group members) were also examined. A meta-analytic approach was chosen to summarize the findings in the microaggression literature. Several search terms and databases were used to identify articles for inclusion. After review, a total of 141 articles with 154 samples contributed effect sizes to our analyses. The results showed that microaggressions were negatively related to psychological well-being and physical health and positively related to coping. The pattern of results was generally the same regardless of the microaggression target, the year the study was conducted, the publication status of the paper, the occupation of the sample, and whether the sample included nonstigmatized groups members or not. This meta-analysis demonstrates the stable, harmful effects associated with experiencing microaggressions. Specifically, microaggressions predicted negative outcomes across individuals and contexts. Thus, actions should be taken to decrease their prevalence within educational and occupational settings.

The D-serine biosynthetic enzyme serine racemase is expressed by reactive astrocytes in the amygdala of human and a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized behaviorally by cognitive deterioration and emotional disruption, and neuropathologically by amyloid-ß (A ß) plaques, neurofibrillary tangles, and complement C3 (C3)-expressing neurotoxic, reactive astrocytes. We previously demonstrated that C3 + reactive astrocytes in the hippocampus and entorhinal cortex of AD patients express serine racemase (SR), which produces the N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine. We show here that C3 + reactive astrocytes express SR in the amygdala of AD patients and in an amyloid mouse model of familial AD (5xFAD). 5xFAD mice also have deficits in cue fear memory recall that is dependent on intact amygdala function. Our results suggest that D-serine produced by reactive astrocytes in the amygdala could contribute to glutamate excitotoxicity and neurodegeneration observed with AD progression.

Inhibition of glial D-serine release rescues synaptic damage after brain injury.

Synaptic damage is one of the most prevalent pathophysiological responses to traumatic CNS injury and underlies much of the associated cognitive dysfunction; however, it is poorly understood. The D-amino acid, D-serine, serves as the primary co-agonist at synaptic NMDA receptors (NDMARs) and is a critical mediator of NMDAR-dependent transmission and synaptic plasticity. In physiological conditions, D-serine is produced and released by neurons from the enzymatic conversion of L-serine by serine racemase (SRR). However, under inflammatory conditions, glial cells become a major source of D-serine. Here, we report that D-serine synthesized by reactive glia plays a critical role in synaptic damage after traumatic brain injury (TBI) and identify the therapeutic potential of inhibiting glial D-serine release though the transporter Slc1a4 (ASCT1). Furthermore, using cell-specific genetic strategies and pharmacology, we demonstrate that TBI-induced synaptic damage and memory impairment requires D-serine synthesis and release from both reactive astrocytes and microglia. Analysis of the murine cortex and acutely resected human TBI brain also show increased SRR and Slc1a4 levels. Together, these findings support a novel role for glial D-serine in acute pathological dysfunction following brain trauma, whereby these reactive cells provide the excess co-agonist levels necessary to initiate NMDAR-mediated synaptic damage.

Forebrain expression of serine racemase during postnatal development.

N-methyl-D-aspartate receptors (NMDARs) are important for synaptogenesis, synaptic maturation and refinement during the early postnatal weeks after birth. Defective synapse formation or refinement underlie cognitive and emotional abnormalities in various neurodevelopmental disorders (NDDs), including schizophrenia (Sz) and autism spectrum disorder (ASD). Serine racemase (SR) is a neuronal enzyme that produces D-serine, a co-agonist required for full NMDAR activation. NMDAR hypofunction as a result of genetic SR elimination and reduced synaptic availability of D-serine reduces neuronal dendritic arborization and spine density. In adult mouse brain, the expression of SR parallels that of NMDARs across forebrain regions including the striatum, amygdala, hippocampus, and medial prefrontal cortex (mPFC). However, there have yet to be studies providing a detailed characterization of the spatial and temporal expression of SR during early periods of synaptogenesis. Here, we examined the postnatal expression of SR in cortical and subcortical brain regions important for learning, memory and emotional regulation, during the first four weeks after birth. Using dual-antigen immunofluorescence, we demonstrate that the number of SR+ neurons steadily increases with postnatal age across the mPFC, amygdala, hippocampus and striatum. We also identified differences in the rate of SR protein induction both across and within brain regions. Analyzing existing human post-mortem brain in situ data, there was a similar developmental mRNA expression profile of SRR and GRIN1 (GluN1 subunit) from infancy through the first decade of life. Our findings further support a developmental role for D-serine mediated NMDAR activation regulating synaptogenesis and neural circuit refinement, which has important implications for the pathophysiology of Sz and other NDDs.

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Targeting lipoic acid to mitochondria: synthesis and characterization of a triphenylphosphonium-conjugated alpha-lipoyl derivative.

Lipoic acid (LA) is a widely used antioxidant that protects mitochondria from oxidative damage in vivo. Much of this protection is thought to be due to the reduction of LA to dihydrolipoic acid (LAH(2)). This reduction is catalyzed in vivo by thioredoxin, thioredoxin reductase (TrxR), and lipoamide dehydrogenase. We hypothesized that specifically targeting LA to mitochondria, the site of most cellular reactive oxygen species production, would make it a more effective antioxidant. To do this, we made a novel molecule, MitoLipoic acid, by attaching lipoic acid to the lipophilic triphenylphosphonium cation. MitoL was accumulated rapidly within mitochondria several-hundred fold driven by the membrane potential. MitoL was reduced to the active antioxidant dihydroMitoLipoic acid by thioredoxin and by lipoamide dehydrogenase but not by TrxR. In isolated mitochondria or cells MitoL was only slightly reduced (5-10%), while, in contrast, LA was extensively reduced. This difference was largely due to the reaction of LA with TrxR, which did not occur for MitoL. Furthermore, in cells MitoL was quantitatively converted to an S-methylated product. As a consequence of its lack of reduction, MitoL was not protective for mitochondria or cells against a range of oxidative stresses. These results suggest that the protective action of LA in vivo may require its reduction to LAH(2) and that this reduction is largely mediated by TrxR.

Targeting dinitrophenol to mitochondria: limitations to the development of a self-limiting mitochondrial protonophore.

The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex.

In Saccharomyces cerevisiae 6 closely related proteins (Did2p, Vps2p, Vps24p, Vps32p, Vps60p, Vps20p) form part of the extended ESCRT III complex. This complex is required for the formation of multivesicular bodies and the degradation of internalized transmembrane receptor proteins. In contrast the human genome encodes 10 homologous proteins (CHMP1A (approved gene symbol PCOLN3), 1B, 2A, 2B, 3 (approved gene symbol VPS24), 4A, 4B, 4C, 5, and 6). In this study we have performed a series of protein interaction experiments to generate a more comprehensive picture of the human CHMP protein-interaction network. Our results describe novel interactions between known components of the human ESCRT III complex and identify a range of putative binding partners, which may indicate new ways in which the function of human CHMP proteins may be regulated. In particular, we show that two further MIT domain-containing proteins (AMSH/STAMBP and LOC129531) interact with multiple components of the human ESCRT III complex.

Glutathionylation of mitochondrial proteins.

Many proteins contain free thiols that can be modified by the reversible formation of mixed disulfides with low-molecular-weight thiols through a process called S-thiolation. As the majority of these modifications result from the interaction of protein thiols with the endogenous glutathione pool, protein glutathionylation is the predominant alteration. Protein glutathionylation is of significance both for defense against oxidative damage and in redox signaling. As mitochondria are at the heart of both oxidative damage and redox signaling within the cell, the glutathionylation of mitochondrial proteins is of particular importance. Here we review the mechanisms and physiological significance of the glutathionylation of mitochondrial thiol proteins.

Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic. Insights into the interaction of ebselen with mitochondria.

Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.

The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B.

Pure hereditary spastic paraplegia is characterized by length-dependent degeneration of the distal ends of long axons. Mutations in spastin are the most common cause of the condition. We set out to investigate the function of spastin using a yeast two-hybrid approach to identify interacting proteins. Using full-length spastin as bait, we identified CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex, as a binding partner. Several different approaches confirmed the physiological relevance of the interaction in mammalian cells. Epitope-tagged CHMP1B and spastin showed clear cytoplasmic co-localization in Cos-7 and PC12 cells. CHMP1B and spastin interacted specifically in vitro and in vivo in beta-lactamase protein fragment complementation assays, and spastin co-immunoprecipitated with CHMP1B. The interaction was mediated by a region of spastin lying between residues 80 and 196 and containing a microtubule interacting and trafficking domain. Expression of epitope-tagged CHMP1B in mammalian cells prevented the development of the abnormal microtubule phenotype associated with expression of ATPase-defective spastin. These data point to a role for spastin in intracellular membrane traffic events and provide further evidence to support the emerging recognition that defects in intracellular membrane traffic are a significant cause of motor neuron pathology.

Solution structure of the Kaposi's sarcoma-associated herpesvirus K3 N-terminal domain reveals a Novel E2-binding C4HC3-type RING domain.

RING domains are found in a large number of eukaryotic proteins. Most function as E3 ubiquitin-protein ligases, catalyzing the terminal step in the ubiquitination process. Structurally, these domains have been characterized as binding two zinc ions in a stable cross-brace motif. The tumorigenic human gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus encodes a ubiquitin-protein ligase termed K3, which functions as an immune evasion molecule by ubiquitinating major histocompatibility complex class I. K3 possesses at its N terminus a domain related to cellular RING domains but with an altered zinc ligand arrangement. This domain was initially characterized as a plant homeodomain, a structure not previously known to function as an E3. Here, it is conclusively demonstrated that the K3 N-terminal domain is a variant member of the RING domain family and not a plant homeodomain. The domain is found to interact with the cellular ubiquitin-conjugating enzymes UbcH5A to -C and UbcH13, which dock to the equivalent surface as on classical cellular RING domains. Interaction with UbcH13 suggests a possible role for K3 in catalyzing Lys(63)-linked ubiquitination.

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE.

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.

Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region.

High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.

A novel gene encoding a coiled-coil mitochondrial protein located at the telomeric end of the human MHC Class III region.

The human Major Histocompatibility Complex (MHC) Class III region, which lies in between the MHC Class I and Class II regions on chromosome 6p21.3, contains approximately 60 genes with diverse functions. Using bioinformatics analyses, we identified a novel open reading frame (ORF) in this region, telomeric of BAT1, which we called Mitochondrial Coiled-Coil Domain 1 (MCCD1). The expression of the predicted ORF in a number of human tissues was confirmed by RT-PCR analysis. An orthologue of the MCCD1 gene was identified in the swine MHC in an analogous position, adjacent to pig BAT1. The overall sequence identity between the human and pig MCCD1 proteins is only 65.9%, but their C-terminal domains are highly conserved, showing 92% identity over 53 residues. The MCCD1 gene encodes a short polypeptide (119 amino acids) which contains a putative coiled-coil domain at its highly conserved C terminus and a predicted mitochondrial localisation signal at its N terminus. Transient expression in mammalian cells of MCCD1 fused at its C terminus to either EGFP or the T7-epitope tag showed that this protein is indeed targeted to mitochondria. Finally, we characterised the polymorphism in this gene using denaturing high-performance liquid chromatography (DHPLC) analysis and found that the MCCD1 gene is highly polymorphic containing an average of 1 single nucleotide polymorphism (SNP) every 99 bp. Interestingly, MCCD1 contains four SNPs within the coding region, three of which cause nonsynonymous and nonconservative changes in the amino acid sequence.

A distinct bipartite motif is required for the localization of inhibitory kappaB-like (IkappaBL) protein to nuclear speckles.

The inhibitory kappaB (IkappaB)-like (IkappaBL) gene is located within the Class III region of the MHC on human chromosome 6. Previous analysis of the predicted amino acid sequence of the human IkappaBL protein revealed three putative functional domains; 2-3 ankyrin repeat sequences, which are similar to the second and third ankyrin repeats of the nuclear factor kappaB (NF-kappaB) protein; three PEST sequence motifs (a sequence that is rich in proline, serine, aspartic acid and threonine residues), which are also found in other IkappaB family members; and a C-terminal leucine zipper-like motif. In the present study we have identified a novel bipartite motif, which is required for nuclear localization of the IkappaBL protein. Analyses of IkappaBL-specific transcripts revealed the existence of a widely expressed spliced variant form of IkappaBL (IkappaBLsv1), which lacks the amino acid sequence GELEDEWQEVMGRFE (where single-letter amino-acid notation has been used). Interestingly, translation of IkappaBL mRNA in vivo was found to initiate predominantly from the second available methionine, thereby resulting in the disruption of the predicted N-terminal PEST sequence. Also, transient expression of T7 epitope-tagged IkappaBL and IkappaBLsv1 proteins in mammalian cells showed that both proteins were targeted to the nucleus, where they accumulate in nuclear speckles. To define the protein domains required for nuclear import and subnuclear localization, a complementary set of deletion mutants and enhanced green fluorescent protein-IkappaBL domain fusions were expressed in mammalian cells. Data from these experiments show that a combination of the ankyrin-repeat region and an adjacent arginine-rich sequence are necessary and sufficient for both nuclear import and speckle localization.