Cytoskeletal protein abnormalities in patients with olivopontocerebellar atrophy — an immunocytochemical and Gallyas silver impregnation study

A highly sensitive silver technique for glial cytoplasmic inclusions (GCI) in olivopontocerebellar atrophy (OPCA) was applied to tissues from 15 patients with neurodegenerative disorders including OPCA, Joseph disease, Alzheimer's disease (AD), Huntington's chorea, Pick disease and three control non-neurological subjects. Brain tissue from both OPCA and AD impregnated positively. Neurons, astroglia and oligodendroglia in the putamen, pontine nucleus and inferior olivary nucleus all impregnated in addition to white matter oligodendroglia. Neuronal inclusions in the pontine nucleus appeared as compact or fibrillary masses, and GCI-bearing oligodendroglia and astrocytes showed homogeneously impregnated somata. The myelinated pontocerebellar tract and the white matter surrounding the inferior olivary nucleus contained a small number of impregnated nerve fibres with a hollow structure, which resembled the myelin sheath. Immunocytochemical studies to clarify these argyrophilic structures in the OPCA subjects employed paired helical filament (PHF), microtubule associated proteins (MAPs), MAP1, MAP2, MAP5, tau, ubiquitin, neurofilament (200 or 70 kilodaltons) and myelin basic protein (MBP) antisera. GCI-bearing white matter oligodendroglia expressed PHF, tau, MAP5 and ubiquitin immunoreactives and non-argyrophilic astroglia were positive for MAP5 antiserum alone. In the putamen, pontine nuclei and inferior olivary nuclei, impregnated neurons as well as the GCI-bearing oligodendroglia immunostained with PHF, tau, MAP5 and ubiquitin antisera and impregnated astroglia were also immunoreactive to these antisera except for being tau negative in the putamen. Silver impregnated nerve fibres showed only MBP immunoreactivity. These findings indicate that the argyrophilia in the OPCA subjects closely correlates with PHF and tau immunoreactivities.     

Comparative immunohistochemical study on the expression of αB crystallin, ubiquitin and stress-response protein 27 in ballooned neurons in various disorders

This report deals with a comparative study on the expression of alpha B crystallin, ubiquitin, stress-response protein 27 (srp 27), srp 72 and phosphorylated neurofilament protein (pNFP) by ballooned neurons in Pick's disease, Creutzfeldt-Jakob disease (CJD), amyotrophic lateral sclerosis (ALS), leptomeningeal carcinomatosis, anterior spinal artery syndrome and pellagra. Immunohistochemical techniques were used. alpha B Crystallin was expressed by the majority of ballooned neurons of Pick's disease and CJD, but not by those of the other disorders. Ubiquitin and srp 27 expression was also restricted to abnormal neurons of Pick's disease and CJD, but the proportion of stained cells was less than that expressing alpha B-crystallin. There was no evidence of ballooned neurons expressing srp 72. Except for those of pellagra patients, phosphorylated neurofilament protein (pNFP) was detected in most abnormal neurons. Our results suggest that the mechanisms involved in formation and maintenance of swollen neurons in Pick's disease and CJD may be different than those of ballooned neurons in the other entities studied.     

Progranulin null mutations in both sporadic and familial frontotemporal dementia

Frontotemporal dementia (FTD) is the second most frequent type of neurodegenerative dementias. Mutations in the progranulin gene (GRN, PGRN) were recently identified in FTDU-17, an FTD subtype characterized by ubiquitin-immunoreactive inclusions and linkage to chromosome 17q21. We looked for PGRN mutations in a large series of 210 FTD patients (52 familial, 158 sporadic) to accurately evaluate the frequency of PGRN mutations in both sporadic and familial FTD, and FTD with associated motoneuron disease (FTD-MND), as well as to study the clinical phenotype of patients with a PGRN mutation. We identified nine novel PGRN null mutations in 10 index patients. The relative frequency of PGRN null mutations in FTD was 4.8% (10/210) and 12.8% (5/39) in pure familial forms. Interestingly, 5/158 (3.2%) apparently sporadic FTD patients carried a PGRN mutation, suggesting the possibility of de novo mutations or incomplete penetrance. In contrast, none of the 43 patients with FTD-MND had PGRN mutations, supporting that FTDU-17 and FTD-MND are genetically distinct. The clinical phenotype of PGRN mutation carriers was particular because of the wide range in onset age and the frequent occurrence of early apraxia (50%), visual hallucinations (30%), and parkinsonism (30%) during the course of the disease. This study supports that PGRN null mutations represent a more frequent cause of FTD than MAPT mutations (4.8% vs. 2.9%) but are not responsible for FTD-MND. It also demonstrates that half of the patients with a PGRN mutation in our series had no apparent family history of dementia. Taking this into account, genetic testing should be now considered more systematically, even in patients without obvious familial history of FTD.     

Cloaked in ubiquitin,a killer hides in plain sight: the molecular regulation of RIPK1

In the past decade, studies have shown how instrumental programmed cell death (PCD) can be in innate and adaptive immune responses. PCD can be a means to maintain homeostasis, prevent or promote microbial pathogenesis, and drive autoimmune disease and inflammation. The molecular machinery regulating these cell death programs has been examined in detail, although there is still much to be explored. A master regulator of programmed cell death and innate immunity is receptor-interacting protein kinase 1 (RIPK1), which has been implicated in orchestrating various pathologies via the induction of apoptosis, necroptosis, and nuclear factor-κB-driven inflammation. These and other roles for RIPK1 have been reviewed elsewhere. In a reflection of the ability of tumor necrosis factor (TNF) to induce either survival or death response, this molecule in the TNF pathway can transduce either a survival or a death signal. The intrinsic killing capacity of RIPK1 is usually kept in check by the chains of ubiquitin, enabling it to serve in a prosurvival capacity. In this review, the intricate regulatory mechanisms responsible for restraining RIPK1 from killing are discussed primarily in the context of the TNF signaling pathway and how, when these mechanisms are disrupted, RIPK1 is free to unveil its program of cellular demise.     

Role of RAG1 autoubiquitination in V(D)J recombination

The variable domains of Ig and T-cell receptor genes in vertebrates are assembled from gene fragments by the V(D)J recombination process. The RAG1–RAG2 recombinase (RAG1/2) initiates this recombination by cutting DNA at the borders of recombination signal sequences (RSS) and their neighboring gene segments. The RAG1 protein is also known to contain a ubiquitin E3 ligase activity, located in an N-terminal region that is not strictly required for the basic recombination reaction but helps to regulate recombination. The isolated E3 ligase domain was earlier shown to ubiquitinate one site in a neighboring RAG1 sequence. Here we show that autoubiquitination of full-length RAG1 at this specific residue (K233) results in a large increase of DNA cleavage by RAG1/2. A mutational block of the ubiquitination site abolishes this effect and inhibits recombination of a test substrate in mouse cells. Thus, ubiquitination of RAG1, which can be promoted by RAG1’s own ubiquitin ligase activity, plays a significant role in governing the level of V(D)J recombination activity.V(D)J recombination plays a central role in the production of antigen receptors by recombining V, D, and J gene segments from their genomic clusters to give rise to the highly varied populations of immunoglobulins and T-cell receptors (1). Recombination starts with the introduction of double-strand breaks by the RAG1/RAG2 protein complex at a pair of recombination signal sequences (RSS) (2, 3), distinguished by the length of the spacer DNA separating their conserved heptamer and nonamer elements. Recombination requires one RSS with a 12-base pair spacer and another with a 23-base pair spacer. Each pair of breaks is then processed by the nonhomologous DNA end-joining group of proteins to produce a junction of two segments of coding sequence (a coding joint) and a junction of the two RSSs (a signal joint) (4). The purified RAG1/2 protein complex displays the correct specificity for pairs of RSSs (5, 6), and has thus been used as a model for the initiation of V(D)J recombination. Until recently, the RAG proteins used for these studies have generally been minimal “core” regions of RAG1 and RAG2 (amino acids 384–1,008 of 1,040 in mouse RAG1 and 1–387 of 527 in RAG2), which are sufficient for specific binding and cleavage activity in a purified cell-free system. Ectopic expression of these truncated proteins supports V(D)J recombination in suitable cell lines, although with differences from the full-length proteins that will be discussed here.A complex composed of core RAG1 and RAG2 is more active than its full-length counterpart in cleavage of extrachromosomal substrates in a hamster cell line, but overall recombination is reported to be lower (7), indicating a defect in the stages of recombination subsequent to DNA cleavage. Similarly, mice or pre-B cells missing the RAG2 C-terminal noncore region are defective in the V to DJ recombination step of Ig heavy chain joining, although the earlier D to J joining step is normal (8). The mice also display an increased prevalence of lymphomas (9). A plant homeo domain (PHD) within the RAG2 C terminus is known to bind to chromatin, and specifically to histone 3 trimethylated on lysine 4 (H3K4me3), which is presumably an important step in directing RAG1/2 to loci bearing this “activating” modification (10). The lack of this domain may largely explain the defective functions of the RAG2 core protein. Similarly, although core RAG1 can support D to J rearrangement at the Ig heavy chain locus in RAG1^−/− pro-B cells, the level is reduced compared with that of full-length RAG1 (FLRAG1) (11), and deletions of certain smaller regions within the RAG1 N terminus have even greater effects (11). Some naturally occurring truncations of the RAG1 N terminus lead to human immunodeficiency (12). The functions of the parts of RAG1 and RAG2 outside of the catalytically essential cores have been reviewed (13). There is also evidence that the RAG1 and RAG2 C termini interact: DNA cleavage by RAG1/2 combinations containing both regions was greatly reduced but was restored upon addition of an H3K4me3-containing peptide (14). Relief of this autoinhibition may synergize with the chromatin-binding effect of the PHD domain to target recombination to the appropriate loci.The significant modulation of recombination in cells, and/or of DNA cleavage in vitro, by these “dispensable” regions of both RAG1 and RAG2 is further modified by covalent modifications of the proteins, which affect their stability or activity. RAG2 becomes phosphorylated at a specific site in its C terminus (T490) at the G1/S stage of the cell cycle, and is then ubiquitinated by the Skp2-SCF ubiquitin ligase, a central regulator of cell cycle progression, leading to its degradation in S phase (15, 16). Phosphorylation of RAG1 at residue S528 by the AMP-dependent protein kinase has also been described (17), in this case leading to increased activity of RAG1/2 both for cell-free DNA cleavage and for recombination in cells.The N terminus of RAG1 contains a Zn-binding motif (amino acids 264–389) that includes a C₃HC₄ RING (really interesting new gene) finger motif closely associated with an adjacent C₂H₂ Zn finger. This domain was shown to have ubiquitin ligase (E3) activity (18, 19), a common feature of RING finger domains, when combined with ubiquitin, the ubiquitin-activating (E1) enzyme, and an appropriate ubiquitin-conjugating (E2) enzyme. A naturally occurring human mutation in this RING finger motif (C328Y) was found to cause the primary immunodeficiency disease Omenn’s syndrome (20). A study of the equivalent mutation in mouse RAG1 (C325Y) showed that it greatly reduced recombination of an extrachromosomal plasmid, as did mutation of the neighboring residue (P326G) (21). Other RING finger residues critical for ubiquitin ligase activity appeared to contribute to robust recombination of extrachromosomal substrates (22). In biochemical experiments carried out with an N-terminal fragment of RAG1 (residues 218–389), the principal site of autoubiquitination was found to be a residue neighboring the RING finger, K233; mutation of this residue (K233M) essentially abolished autoubiquitination of the fragment (18).In this article, we assess the site or sites and extent of autoubiquitination of RAG1, the consequences of this modification for RAG1/RAG2 activity in a cell-free system and in cells, and the functional relationship between this modification and the histone-recognizing PHD domain of RAG2. We prepare FLRAG1 in complex with either full-length RAG2 (FLRAG2) or core RAG2 and find that FLRAG1 undergoes autoubiquitination specifically at K233. The ubiquitination of RAG1 protein enhances coupled cleavage by the RAG1/RAG2 complex of a 12/23 RSS pair by about fivefold. RAG1 autoubiquitination also ap-pears to be important for supporting V(D)J recombination in cells.     

小鼠睾丸组织特异表达基因Rnf148的鉴定及其编码E3泛素连接酶的功能分析

目的 研究小鼠Rnf148基因表达的时空特异性及其环指结构域的E3泛素连接酶功能。 方法 提取不同成年小鼠组织、不同胚胎期组织和出生后小鼠睾丸组织的总RNA,通过实时荧光RT-PCR和Northern杂交分析小鼠Rnf148基因的表达谱。构建包含整个Rnf148蛋白的环指结构域与谷胱甘肽-S-转移酶（GST）的融合蛋白原核表达载体,在BL21细菌中诱导表达后,经GST琼脂糖凝胶纯化GST-Rnf148重组蛋白。体外泛素化反应试验检测GST-Rnf148重组蛋白的E3泛素连接酶功能。 结果 在小鼠13种不同器官组织中,Rnf148 mRNA仅存在于睾丸组织中。进一步Northern杂交验证了只在小鼠睾丸组织表达一个1.2 kb左右的Rnf148基因mRNA片段。小鼠Rnf148基因在胚胎期及出生后3周内不表达,出生后21 d开始表达,25 d后达到表达高峰并一直持续表达。实验成功诱导表达并纯化了GST-Rnf148重组蛋白,体外蛋白泛素化反应显示该重组蛋白具有E3泛素连接酶的功能。 结论 小鼠Rnf148基因特异地表达在出生3周后的睾丸组织中,Rnf148蛋白的环指结构域具有泛素连接酶活性。     

Apolipoprotein B100 quality control and the regulation of hepatic very low density lipoprotein secretion

Apolipoprotein B （apoB） is the main protein component of very low density lipoprotein （VLDL） and is necessary for the assembly and secretion of these triglyceride （TG）-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein （LDL） in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation （ERAD） by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regu- lated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autop- hagy and postprandial activation of the unfolded protein response （UPR） may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.     

SUMO enhances unfolding of SUMO–polyubiquitin-modified substrates by the Ufd1/Npl4/Cdc48 complex

The Ufd1/Npl4/Cdc48 complex is a universal protein segregase that plays key roles in eukaryotic cellular processes. Its functions orchestrating the clearance or removal of polyubiquitylated targets are established; however, prior studies suggest that the complex also targets substrates modified by the ubiquitin-like protein SUMO. Here, we show that interactions between Ufd1 and SUMO enhance unfolding of substrates modified by SUMO–polyubiquitin hybrid chains by the budding yeast Ufd1/Npl4/Cdc48 complex compared to substrates modified by polyubiquitin chains, a difference that is accentuated when the complex has a choice between these substrates. Incubating Ufd1/Npl4/Cdc48 with a substrate modified by a SUMO–polyubiquitin hybrid chain produced a series of single-particle cryo-EM structures that reveal features of interactions between Ufd1/Npl4/Cdc48 and ubiquitin prior to and during unfolding of ubiquitin. These results are consistent with cellular functions for SUMO and ubiquitin modifications and support a physical model wherein Ufd1/Npl4/Cdc48, SUMO, and ubiquitin conjugation pathways converge to promote clearance of proteins modified with SUMO and polyubiquitin.

Ubiquitin and small ubiquitin-like modifier (SUMO) conjugation represent two essential post-translational modifications that participate in nearly every cellular process (1–4). Substrate conjugation by ubiquitin and ubiquitin-like proteins such as SUMO requires the sequential activities of three-enzyme cascades involving E1 activating enzymes, E2 conjugating enzymes, and E3 protein ligases that result in covalent modification of targets, principally on substrate lysine residues. These priming conjugation events can be further remodeled by ligases and proteases to generate ubiquitin and ubiquitin-like polymeric chains with different topologies (4), each of which has the potential to signal through factors that recognize different chain topologies. Several studies suggest overlap in ubiquitin and SUMO conjugation pathways in various cellular processes including heat shock and DNA damage responses and the maintenance of subcellular structures including promyelocytic leukemia or PML bodies (5–11).SUMO-targeted ubiquitin ligases (STUbLs) represent a conserved class of E3 ubiquitin ligases that target poly-SUMO-conjugated proteins for modification by ubiquitin (12, 13). While STUbLs can target proteins in the absence of SUMO (14), specificity for SUMO chains is achieved by E3 ligase subunits that contain tandem SUMO Interaction Motifs (SIMs) (9, 15). Key phenotypes of STUbL dysfunction include genomic instability, hypersensitivity to genotoxins, and accumulation of high molecular weight SUMO chains (16, 17). Several lines of evidence suggest that STUbLs provide a means to clear SUMO-conjugated proteins after events such as heat shock or DNA damage (18–20). Biochemically, STUbL-mediated ubiquitin conjugation of SUMO-modified targets can result in dual modification with SUMO and ubiquitin on different lysines in the target complex, or hybrid chain modification with polyubiquitin conjugated to a SUMO chain. Proteomics studies revealed the presence of ubiquitin-conjugated SUMO in vivo, lending support to the idea that hybrid chains could serve as intermediates in protein clearance (21, 22).Potential readers of substrates modified with SUMO–polyubiquitin hybrid chains include RAP80 in the mammalian DNA damage response (23) and the Ufd1/Npl4/Cdc48 complex in budding and fission yeast (24, 25). The Ufd1/Npl4/Cdc48 complex is a crucial component of ubiquitin-mediated protein metabolism as the universal segregase that removes targets marked with polyubiquitin from complexes and membranes. It is composed of the Ufd1/Npl4 dimer adaptor, which determines substrate specificity, and the Cdc48 hexamer, a AAA+ protein that couples adenosine triphosphate (ATP) hydrolysis to protein unfolding (26–28). Recent structural studies have demonstrated that lysine 48-linked polyubiquitin acts as a recruitment signal and the initiation site for unfolding by the complex, as a peptide corresponding to unfolded ubiquitin was observed threaded across the Npl4 surface and into the channel formed by the Cdc48 hexamer (28). Subsequent studies revealed a complex interplay between interactions with polyubiquitylated substrates and productive unfolding, namely that Ufd1/Npl4/Cdc48 can unfold any ubiquitin within the K48-linked chain, but productive substrate unfolding only occurs after unfolding a ubiquitin molecule that is proximal and N-terminal but not C-terminal to the substrate (29). These studies suggest that the topology of substrate and polyubiquitin defines whether a substrate can be unfolded and that the search for substrate proximal ubiquitin may be a rate-limiting step.Ufd1 in budding yeast possesses a C-terminal SIM that was uncovered in the anti-recombinogenic helicase Srs2, where the SIM plays a critical role in the recognition of SUMO-modified proliferating cell nuclear antigen (30, 31). The analogous C-terminal SIM in fission yeast Ufd1 interacts with SUMO and contributes to maintenance of genome integrity (24). Colocalization of Ufd1 to SUMO foci increases during the DNA damage response, and the resolution of SUMO foci appears dependent on the C-terminal SIM of Ufd1 as its deletion results in an increase in intensity of DNA damage-associated SUMO foci in fission yeast (32). Furthermore, deletion of the SIM is genetically epistatic with STUbL mutants with respect to the DNA damage response (32). Consistent with these observations, substantial overlap exists in perturbations of the proteome that result from deletion of the Ufd1 C-terminus or disruption of STUbL function, leading to the hypothesis that Ufd1 and STUbLs work in the same pathways (33).Here, we set out to reconstitute substrates modified with SUMO and polyubiquitin to mimic SUMO-modified proteins after STUbL-mediated ubiquitylation. We then used these substrates to determine their propensities for unfolding by the Saccharomyces cerevisiae Ufd1/Npl4/Cdc48 complex to define contributions of SUMO to this process. We show that compared to the canonical polyubiquitin-only-modified substrates, the Ufd1/Npl4/Cdc48 complex preferentially unfolds SUMO–polyubiquitin dual-modified substrates in a Ufd1 SIM and SUMO-dependent manner. This SUMO-dependent unfolding activity is also conserved in the Schizosaccharomyces pombe Ufd1/Npl4/Cdc48 complex. Additionally, we present previously unreported single-particle cryogenic electron microscopy (cryo-EM) structures of the Ufd1/Npl4/Cdc48 complex with a SUMO–polyubiquitin substrate in multiple states showing the complex prior to and during ubiquitin unfolding. Our results support a model in which SUMO enhances unfolding by increasing interactions with the substrate, potentially facilitating the search for substrate proximal ubiquitin.