A large open reading frame ( orf1995 ) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival. Received: 18 August 1996 / Accepted: 24 September 1996     

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G₄C₂ repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G₄C₂ RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Structural and biochemical characterisation of the C9orf72-SMCR8 complex sheds light on its overall architecture and highlights its role as a multi-functional scaffold for coordinating autophagy.     

The genomic structure of C14orf1 is conserved across eukarya

We have recently cloned the gene C14orf1, which is strongly expressed in normal testis and in several cancer cell lines and tumors. This gene maps to 14q24.3 and is interrupted by four introns. Two of them are also represented in the open reading frame of Schizosaccharomyces pombe in the same phase. In Arabidopsis taliana only the first of the two introns was found, in the same phase as the corresponding ones in S. pombe and human. Disruption of the ortholog in Saccharomyces cerevisiae (Yer044c) led to a severe growth defect, and C14orf1 failed to complement mutant yeast when put under the control of the natural Yer044c promoter. Further studies are needed to understand the causes underlying the high degree of conservation of the C14orf1 genomic structure. Received: 7 February 2000 / Accepted: 14 April 2000     

Intra- and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cytoplasm from Raphanus sativus and Raphanus raphanistrum

In order to gain a better understanding of the evolution of Ogura male-sterile cytoplasm in radish, a large-scale sequence analysis of mitochondrial orf138 was conducted using 107 Japanese wild radishes, 29 cultivated radishes and seven Raphanus raphanisturum. A single approximately 0.8-kb fragment containing the orf138 locus was amplified from each plant by PCR, and the nucleotide sequence of an entire coding region of orf138 was determined by direct-sequencing procedures. An identical sequence to the published orf138 (Type A) was identified in Japanese wild radish, including a single plant in a population near Kagoshima prefecture where Ogura (1968) first found ’Ogura male-sterile radish’. Thus, it was confirmed that the ’Ogura male-sterile cytoplasm’ was derived from Japanese wild radish, with a Type A orf138 sequence, growing in this area. A total of six nucleotide changes and a single insertion/deletion (indel) were found in orf138 from both wild and cultivated radishes. By a combination of mutations, the orf138 sequences of the 143 radish plants were classified into nine types. Based on the pattern of mutations and the distribution of orf138 variants, it was concluded that the orf138 variants are derived from Type B or C, after Ogura-type cytoplasm was introduced from R. raphanistrum into Japanese wild radish. Received: 19 December 2000 / Accepted. 26 January 2001     

Positive selection drives lactoferrin evolution in mammals

Lactoferrin (LF) is a member of the transferrin family that is abundantly expressed and secreted by glandular epithelial cells. The biological functions of LF involve in iron homeostasis regulation of the body and antibacterial activity. Previous studies demonstrated that it had a high cationic N-terminal domain that could interact with glycosaminoglycans, lipopolysaccharides and the bacterial virulence protein. Two anti-microbial peptides, lactoferricin (LFcin) and lactoferrampin (LFampin), were also isolated and identified in N-terminal of LF. Although the antibacterial mechanism was carefully studied, little was known about the molecular evolution of LF. In this study, we estimated the nonsynonymous-to-synonymous substitution ratios ( w = d_N \mathord

Late‐onset progressive retinal atrophy in the Gordon and Irish Setter breeds is associated with a frameshift mutation in C2orf71

Progressive retinal atrophy (PRA) in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 14 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s) responsible have yet to be identified. Using genome‐wide association with 16 Gordon Setter PRA cases and 22 controls, we identified a novel PRA locus, termed rod–cone degeneration 4 (rcd4), on CFA17 (P_raw = 2.22 × 10^?8, P_genome = 2.00 × 10^?5), where a 3.2‐Mb region was homozygous within cases. A frameshift mutation was identified in C2orf71, a gene located within this region. This variant was homozygous in 19 of 21 PRA cases and was at a frequency of approximately 0.37 in the Gordon Setter population. Approximately 10% of cases in our study (2 of 21) are not associated with this C2orf71 mutation, indicating that PRA in this breed is genetically heterogeneous and caused by at least two mutations. This variant is also present in a number of Irish Setter dogs with PRA and has an estimated allele frequency of 0.26 in the breed. The function of C2orf71 remains unknown, but it is important for retinal development and function and has previously been associated with autosomal recessive retinitis pigmentosa in humans.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

Identification,expression and tissue distribution of a renalase homologue from mouse

FAD (flavin adenine dinucleotide)-dependent monoamine oxidases play very important roles in many biological processes. A novel monoamine oxidase, named renalase, has been identified in human kidney recently and is found to be markedly reduced in patients with end-stage renal disease (ESRD). Here, we reported the identification of a renalase homologue from mouse, termed mMAO-C (mouse monoamine oxidase-C) after the monoamine oxidase-A and -B (MAO-A and -B). This gene locates on the mouse chromosome 19C1 and its coding region spans 7 exons. The deuced amino acid sequences were predicted to contain a typical secretive signal peptide and a conserved amine oxidase domain. Phylogenetic analysis and multiple sequences alignment indicated that mMAO-C-like sequences exist in all examined species and share significant similarities. This gene has been submitted to the NCBI GenBank database (Accession number: DQ788834). With expression vectors generated from the cloned mMAO-C gene, exogenous protein was effectively expressed in both prokaryotic and eukaryotic cells. Recombinant mMAO-C protein was secreted out of human cell lines, indicating the biological function of its signal peptide. Moreover, tissue expression pattern analysis revealed that mMAO-C gene is predominantly expressed in the mouse kidney and testicle, which implies that kidney and testicle are the main sources of renalase secretion. Shortly, this study provides an insight into understanding the physiological and biological functions of mMAO-C and its homologues in endocrine.     

C21orf5, a New Member of Dopey Family Involved in Morphogenesis, Could Participate in Neurological Alterations and Mental Retardation in Down Syndrome

Availability of the human genome sequence promises importantprogress in the understanding of human pathologies, particularlyfor multifactorial diseases. Among these, Down syndrome (DS)is the most frequent genetic cause of mental retardation. Acritical region of chromosome 21, the Down syndrome ChromosomalRegion-1 (DCR-1), is responsible for many features of the DSphenotype including mental retardation. We studied DCR-1 C21orf5as a new candidate gene for DS considering its restricted expressionin key brain regions altered in DS patients and involved inlearning and memory processes. To elucidate C21orf5 molecularfunction, we performed a comparative study of protein sequencesin several species and showed that C21orf5 represents a newmember of the Dopey leucine zipper-like family. The C21orf5C-termini contains two highly conserved leucine-like zipperdomains in invertebrate and vertebrate species. Evolution analysisindicated a common ancestral origin of these protein sequencesalso suggesting a conserved function of this gene throughoutphylogenesis. Mutations of the known C21orf5 homologous genesAspergillus nidulans DopA, Saccharomyces cerevisiae Dop1 andCaenorhabditis elegans pad1, determine morphological abnormalities.We studied transgenic mice carrying the human C21orf5 gene andwe showed that this gene is overexpressed in brain regions byin situ hybridization and by real-time RT–PCR experiments.Interestingly, we also showed that these transgenic mice havean increased density of cortical cells overexpressing C21orf5.Similarly, DS patients have an altered lamination pattern intheir cortex. Considering together our and previous findings,we suggest that the human dopey family member, C21orf5, couldplay a role in brain morphogenesis and, when overexpressed,it could participate in neurological features and mental retardationobserved in DS patients.     

C9orf72: At the intersection of lysosome cell biology and neurodegenerative disease

The discovery that expansion of a hexanucleotide repeat within a noncoding region of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia raised questions about C9orf72 protein function and potential disease relevance. The major predicted structural feature of the C9orf72 protein is a DENN (differentially expressed in normal and neoplastic cells) domain. As DENN domains are best characterized for regulation of specific Rab GTPases, it has been proposed that C9orf72 may also act through regulation of a GTPase target. Recent genetic and cell biological studies furthermore indicate that the C9orf72 protein functions at lysosomes as part of a larger complex that also contains the Smith‐Magenis chromosome region 8 (SMCR8) and WD repeat‐containing protein 41 (WDR41) proteins. An important role for C9orf72 at lysosomes is supported by defects in lysosome morphology and mTOR complex 1 (mTORC1) signaling arising from C9orf72 KO in diverse model systems. Collectively, these new findings define a C9orf72‐containing protein complex and a lysosomal site of action as central to C9orf72 function and provide a foundation for the elucidation of direct physiological targets for C9orf72. Further elucidation of mechanisms whereby C9orf72 regulates lysosome function will help to determine how the reductions in C9orf72 expression levels that accompany hexanucleotide repeat expansions contribute to disease pathology.      

The identification and characterization of a novel protein,c19orf10, in the synovium

Joint inflammation and destruction have been linked to the deregulation of the highly synthetic fibroblast-like synoviocytes (FLSs), and much of our current understanding of the mechanisms that underlie synovitis has been collected from studies of FLSs. During a proteomic analysis of FLS cells, we identified a novel protein, c19orf10 (chromosome 19 open reading frame 10), that was produced in significant amounts by these cells. The present study provides a partial characterization of c19orf10 in FLSs, synovial fluid, and the synovium. Murine monoclonal and chicken polyclonal antibodies were produced against recombinant human c19orf10 protein and used to examine the distribution of c19orf10 in cultured FLSs and in synovial tissue sections from patients with rheumatoid arthritis or osteoarthritis. The intracellular staining pattern of c19orf10 is consistent with localization in the endoplasmic reticulum/Golgi distribution. Sections of rheumatoid arthritis and osteoarthritis synovia expressed similar patterns of c19orf10 distribution with perivascular and synovial lining staining. Double-staining in situ analysis suggests that fibroblast-like synovial cells produced c19orf10, whereas macrophages, B cells, or T cells produced little or none of this protein. There is evidence of secretion into the vascular space and the extracellular matrix surrounding the synovial lining. A competitive enzyme-linked immunosorbent assay confirmed the presence of microgram levels of c19orf10 in the synovial fluids of patients with one of various arthropathies. Collectively, these results suggest that c19orf10 is an FLS-derived protein that is secreted into the synovial fluid. However, the significance of this protein in synovial biology remains to be determined. The absence of known structural motifs or domains and its relatively late evolutionary appearance raise interesting questions about its function.     

Systemic deregulation of autophagy upon loss of ALS- and FTD-linked C9orf72

A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Molecular Cloning and Characterization of a Novel Human C4orf13 Gene, Tentatively a Member of the Sodium Bile Acid Cotransporter Family

By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human cDNA (C4orf13). This cDNA is 2706 bp in length, encoding a 340-amino-acid polypeptide that contains a typical SBF (sodium bile acid cotransporter family) domain and ten possible transmembrane segments. The putative protein C4orf13 shows high similarity with its orthologs in Mus musculus and Xenopus laevis. Human C4orf13 is mapped to chromosome 4q31.2 and contains 12 exons. RT-PCR analysis shows that human C4orf13 is widely expressed in human tissues, and the expression levels in liver and lung are relatively high, expression levels in placenta, kidney, spleen, and thymus are moderate, low levels of expression are detected in heart, prostate, and testis.The nucleotide sequence reported in this paper has been deposited to GenBank under accession number AY346324.