Intracellular distribution of the reductive and oxidative pentose phosphate pathways in two diatoms

Diatoms contribute a large proportion to the worldwide primary production and are particularly effective in fixing carbon dioxide. Possibly because diatom plastids originate from a secondary endocytobiosis, their cellular structure is more complex and metabolic pathways are rearranged within diatom cells compared to cells containing primary plastids. We annotated genes encoding isozymes of the reductive and oxidative pentose phosphate pathways in the genomes of the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum and bioinformatically inferred their intracellular distribution. Prediction results were confirmed by fusion of selected presequences to Green Fluorescent Protein and expression of these constructs in P. tricornutum. Calvin cycle enzymes for the carbon fixation and reduction of 3‐phosphoglycerate are present in single isoforms, while we found multiple isoenzymes involved in the regeneration of ribulose‐1,5‐bisphosphate. We only identified one cytosolic sedoheptulose‐1,7‐bisphosphatase in both investigated diatoms. The oxidative pentose phosphate pathway seems to be restricted to the cytosol in diatoms, since we did not find stromal glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconolactone dehydrogenase isoforms. However, the two species apparently possess a plastidic phosphogluconolactonase. A 6‐phosphogluconolactone dehydrogenase is apparently plastid associated in P. tricornutum and might be active in the periplastidic compartment, suggesting that this compartment might be involved in metabolic processes in diatoms. Abbreviations: AL: aldolase, ATP: adenosine triphosphate, Chl: Chlorophyll, DIC: Normanski differential interference contrast, ER: endoplasmic reticulum, EST: expressed sequence tag, FBA: fructose‐1,6‐bisphosphate aldolase, FBPase: fructose‐1,6‐bisphosphatase, GAPDH: glycerinaldehyd‐3‐phosphate dehydrogenase, GFP: enhanced Green Fluorescent Protein, GPDH: glucose‐6‐phosphate dehydrogenase, GPI: glucose‐6‐phosphate isomerase, HMM: Hidden Markov Models, JGI: Joint Genome Institute, NADPH: nicotinamide adenine dinucleotide phosphate, NN: Neuronal networks, OPP: oxidative pentose phosphate pathway, PCR: Polymerase Chain Reaction, PGDH: 6‐phosphogluconolactone dehydrogenase, PGK: phosphoglycerate kinase, PGL: phosphogluconolactonase, Phatr2: version 2.0 of the Phaeodactylum tricornutum genome, PRK: phosphoribulokinase, RPE: ribulose‐phosphate epimerase, RPI: ribose‐5‐phosphate isomerase, RuBisCO: ribulose‐1,5‐bisphosphate carboxylase/oxygenase, SBPase: sedoheptulose‐1,7‐bisphosphatase, TAL: transaldolase, Thaps3: version 3.0 of the Thalassiosira pseudonana genome, TKL: transketolase, TPI: triosephosphate isomerase, UGGtransferase: UDP glucose‐starch glycosyl transferase. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis

The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA). We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena. Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes. Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont. Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages. Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate. Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria. The data provide evidence for the chimaeric nature of eukaryotic genomes. Received: 20 December 1996 / 5 February 1997     

Complete sequence of the mitochondrial genome of a diatom alga Synedra acus and comparative analysis of diatom mitochondrial genomes

The first two mitochondrial genomes of marine diatoms were previously reported for the centric Thalassiosira pseudonana and the raphid pennate Phaeodactylum tricornutum. As part of a genomic project, we sequenced the complete mitochondrial genome of the freshwater araphid pennate diatom Synedra acus. This 46,657 bp mtDNA encodes 2 rRNAs, 24 tRNAs, and 33 proteins. The mtDNA of S. acus contains three group II introns, two inserted into the cox1 gene and containing ORFs, and one inserted into the rnl gene and lacking an ORF. The compact gene organization contrasts with the presence of a 4.9-kb-long intergenic region, which contains repeat sequences. Comparison of the three sequenced mtDNAs showed that these three genomes carry similar gene pools, but the positions of some genes are rearranged. Phylogenetic analysis performed with a fragment of the cox1 gene of diatoms and other heterokonts produced a tree that is similar to that derived from 18S RNA genes. The introns of mtDNA in the diatoms seem to be polyphyletic. This study demonstrates that pyrosequencing is an efficient method for complete sequencing of mitochondrial genomes from diatoms, and may soon give valuable information about the molecular phylogeny of this outstanding group of unicellular organisms.     

Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation

Summary: The vast majority of the peptides produced during protein degradation by the cytosolic proteasome‐ubiquitin system are consecutively hydrolyzed to single amino acids by multiple cytosolic peptidases preferring intermediate length or short substrates. The small fraction of peptides surviving the aggressive cytosolic environment can be recruited for presentation by major histocompatibility complex (MHC) class I molecules. However, such peptides may frequently have to be adapted to the strict MHC class I‐binding requirements by one or several N–terminal‐trimming steps. A recent model proposes that an initial step, in which peptides of 15 or more residues are shortened by cytosolic tripeptidylpeptidase II, is followed by additional trimming by cytosolic or endoplasmic reticulum (ER) aminopeptidases. In humans, at least two ER resident aminopeptidases, ERAP1 and ERAP2, contribute to trimming of human leukocyte antigen class I ligands. These interferon‐γ‐regulated metallopeptidases show distinct substrate preferences and may have to act in a concerted fashion to remove some complex or longer N‐terminal extensions and to trim the full spectrum of precursor peptides. This task is likely facilitated by the formation of presumably heterodimeric ERAP1‐2 complexes. RNA interference experiments suggest that both enzymes are important for normal antigen presentation, but precise determination of the extent and the cellular context of their requirement will be left to future experimentation.     

Beyond the proteasome: trimming,degradation and generation of MHC class I ligands by auxiliary proteases

The proteasome is now recognized to be implicated in the generation of the vast majority of MHC class I ligands. Moreover, it is probably the only cytosolic protease generating their carboxyterminals. However, solid evidence documents a role of additional and only partly identified proteases in MHC class I antigen processing. Cytosolic tripeptidyl peptidase (TTP II) may be able to carry out some functions normally ascribed to the proteasome, including that of generating antigenic peptides. Several cytosolic enzymes, including bleomycin hydrolase (BH) and puromycin-sensitive aminopeptidase (PSA), but especially the IFNgamma-inducible leucyl aminopeptidase (LAP), can trim the aminoterminal ends of class I ligands. The vast majority of cytosolic peptides is degraded, a process likely to limit antigen presentation, in which thimet oligopeptidase (TOP) may play an important role. Proteolytic activity in the secretory pathway, though much more limited than in the cytosol, also contributes to class I antigen presentation. Signal peptide fragments and peptides at the carboxyterminal end of various proteins targeted to the endoplasmic reticulum can be highly efficient TAP-independent class I ligands. However, an as yet unidentified luminal trimming aminopeptidase may eventually turn out to play the most important role for class I ligand generation in the secretory pathway. Defining the extent of the involvement of cytosolic and luminal peptidases in class I antigen processing will be a challenging task for the future.     

Presentation of exogenous antigens by macrophages: analysis of major histocompatibility complex class I and II presentation and regulation by cytokines

There is an antigen presenting cell (APC) in the lymphoid organs capable of presenting exogenous antigen (Ag) with major histocompatibility complex (MHC) class I molecules. This study was initiated to isolate clones of these APC to definitively establish their phenotype and to further study their properties. Murine bone marrow macrophages (BM MΦ) were immortalized by overexpressing myc and raf oncogenes. Five BM MΦ cell lines were generated that are phagocytic and expressed at their surface MΦ differentiation Ag. All five cell lines processed and presented exogenous ovalbumin (OVA) with MHC class I molecules. They all presented OVA-linked to a phagocytic substrate 10²–10⁴-fold more efficiently than soluble Ag. Clonal isolates of two of the MΦ cell lines had an identical phenotype and functional properties as the uncloned lines. These results definitively establish that MΦ are APC with the capacity of presenting exogenous Ag with MHC class I molecules. Interferon (IFN)-γ interleukin-4, granulocyte-macrophage colony stimulating factor and lipopolysaccharide either alone or in combination induced little or no augmentation and in some cases decreased presentation of exogenous OVA with MHC class I. In contrast, all of MΦ activating factors increased MHC class I expression. Moreover, IFN-γ increased the presentation of cytosolic OVA, demonstrating differences between the presentation of cytosolic Ag versus exogenous Ag with MHC class I. Finally, some lines constitutively processed and presented exogenous OVA with MHC class II while others only presented after stimulation with IFN-γ. These results demonstrate that the pathways involved in the presentation of exogenous Ag with MHC class I and class II are independently regulated and that a cloned cell is capable of presenting exogenous Ag through both pathways.     

Phylogenetic analysis of diatom <Emphasis Type="Italic">coxI</Emphasis> genes and implications of a fluctuating GC content on mitochondrial genetic code evolution

In order to address the relationships among diatom groups and to investigate possible changes in their mitochondrial (mt) genetic codes, we have analyzed a 1.1-kb region of the cytochrome c oxidase subunit I (coxI) gene from eight diverse diatom species. A phylogenetic analysis of these coxI sequences including representative species of the Phaeophyta, Xanthophyta, Eustigmatophyta and Haptophyta showed that the diatoms (Bacillariophyta) formed a well-supported monophyletic group. Of the eight species investigated, four have been classified together as radial centric diatoms based on morphology. However, in our coxI tree, the two radial centrics belonging to the order Thlassiosirales (Skeletonema costatum and Thalassiosira nordenskioldii) were placed as the sister group to the multipolar centric diatoms, while the other two radial centrics (Melosira ambigua and Rhizosolenia setigera) were in another clade. Also, in two species of the Tharassiosirales we found UGA codons that occur at conserved tryptophan (Trp) sites in the coxI sequences, strongly indicating that UGA codes for Trp in these diatoms. No evidence of a deviant genetic code was detected in the other analyzed diatom species. There was no apparent relationship between the nucleotide third-position GC content of mtDNA (based on the sequenced coxI region) and the presence of a deviant genetic code. Received: 11 December 1998 / 30 August 1999     

Expression of β-glucosidase and endo-β-1,4-glucanase during development of Chaetomium fusisporale and their characterization

In the present study, the production of β-glucosidase and endoglucanase and the pattern of their corresponding polymorphic forms occurring in the extracellular and cytosolic filtrates of Chaetomium fusisporale (ATCC 64772) has been studied. When C. fusisporale was grown on carboxymethyl cellulose (CMC) as the sole carbon source, the extracellular fractions contained most of these enzymes. The distribution of the enzymes varied with the age of the culture. By the use of electrophoretic techniques the presence of two forms of β-glucosidase, i.e. β-GLU II and β-GLU III was detected in extracellular and cytosolic filtrates at early stages of growth, while at later stages two more isozymes of β-glucosidase, i.e. β-GLU I and β-GLU IV, only appeared in the cytosolic filtrates. Similary, endoglucases isozymes EG I and EG II were present in both fractions at early stages of growth but at later stages, with the production of perithecia, EG III appeared. Thus, EG III, β-GLU I and β-GLU IV may indicate that they are correlated with the development of the fungus. In order to differentiate further between the multiple molecular forms of these enzymes two of them have been characterized with respect to temperature, pH, ethylenediamine tetra-acetic acid (EDTA) and metal ions.     

Heterogeneity of class I and class II MHC sequences in Schistosoma mansoni

We investigated the genetic variations in class I and class II major histocompatibility complex (MHC) genes of Schistosoma mansoni and the effects of host MHC genotypes. S. mansoni was maintained in combinations of two mouse strains with different MHC genotypes, and the MHC gene sequences of the cercariae were investigated. The detected class I MHC gene sequences were variable, with high similarity between the H-2D^b murine host and the parasite. For other combinations, however, the parasite sequence was homologous to those of anthropoids. All class II MHC sequences detected in S. mansoni were homologous to those of anthropoids. Our results suggest that the genetic variation in the MHC sequences of S. mansoni is derived in part from the current host, indicating horizontal transfer of the sequences from mammal to parasite.     

Heterogeneity of class I and class II MHC sequences in <Emphasis Type="Italic">Schistosoma japonicum</Emphasis> from different endemic regions in mainland China

The present study examined sequence variation in class I and class II major histocompatibility complex (MHC) genes among Schistosoma japonicum isolates from different endemic regions in mainland China and assessed the level of horizontal gene transfer and sequence similarity between parasites and their hosts. S. japonicum cercariae were used to infect male adult rabbits to obtain adult S. japonicum samples. A portion of the class I MHC gene (pMHC I) and class II MHC genes (pMHC II) were amplified separately from individual adult trematodes by polymerase chain reaction and sequenced. Among all the examined isolates of S. japonicum, sequence differences between male and female parasites were 0.0–26.6% for pMHC I and 0.0–7.0% for pMHC II. Sequence variations between male and female parasites among different geographical locations from the mountainous areas were 1.1–26.6% for pMHC I and 1.5–3.0% for pMHC II. Sequence variations between samples from Yunnan and those from Sichuan were 2.7–23.5% for pMHC I and 1.1–3.7% for pMHC II. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0–25.0% for pMHC I and 0.0–7.0% for pMHC II. Sequence variations between S. japonicum isolates from mountainous areas, and those from lake/marshland areas were 0.0–26.1% for pMHC I and 0.4–6.1% for pMHC II. BLASTN analysis indicated that all the pMHC II sequences showed high homology to a portion of exon 3 in rabbit MHC class II DP beta gene with more than 89% similarity, and all the pMHC I sequences except isolates in Yunnan (Eryuan) revealed high homology to the portion of exon 2 in rabbit MHC I gene with more than 81% similarity. Phylogenetic analysis showed no specific clustering comprising parasites from single geographical or endemic regions, and the paired parasites were even found in different clusters. These results demonstrated that pMHC I and II of S. japonicum isolates in mainland China existed heterogeneity, but the pMHC I, II, or combined sequences were not suitable markers for examining genetic relationship among different isolates from endemic regions in mainland China.     

Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-γ) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-γ-induced surface expression of class II antigens as well as the IFN-γ-induced up-regulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-γ for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started ≈ 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E₂, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.     

Expression of MHC class I and class II antigens in primary breast carcinomas and synchronous nodal metastases

Expression of the major histocompatibility (MHC) class I and class II antigens was studied by immunohistochemistry in a series of 70 primary breast carcinomas and in nodal metastases. In particular, the expression of class I (HLA A-B-C) and class II (DP, DQ and DR) molecules was compared in: a) primary breast cancers devoid of nodal metastases (n = 36) and tumors exhibiting metastatic deposits (n = 34) at the time of surgery, and b) primary breast carcinomas and their corresponding synchronous axillary nodal metastases. Reduced or absent HLA A-B-C antigen expression was seen in approximately 54.3% of primary breast carcinomas, whereas a partial or complete induction of class II products was observed in 18.5% (DQ), 30% (DP) or 48.5% (DR) of the same cases. An almost complete overlap of antigen expression was observed in breast tumors in which no metastases were found by histological examination of axillary nodes and in neoplasms showing histologically-diagnosed synchronous metastases. The reactivity for class I and class II antigens in nodal metastases roughly paralleled that exhibited by corresponding primary tumors. A discordant expression was seen in 11 cases (32%) stained for HLA A-B-C and in 8 (24%), 7 (21%) and 6 (18%) cases assayed for DP, DQ and DR products, respectively. When a discordant expression was detected, either decreased or increased staining patterns were observed in metastases. The finding of overlapping MHC antigenic profiles in the majority of primary breast tumors and nodal metastases casts doubts on the hypothesis that loss of MHC antigens can play an important role in the seeding and growth of metastatic breast carcinoma cells.     

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

Summary The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.     

Differential expression of HLA class I and II antigens in primary and metastatic melanomas

Class I and II histocompatibility antigen expression was studied in cryostat sections of biopsy tissues from 15 patients diagnosed as suffering from malignant melanoma, using monoclonal antibodies against HLA class I and II monomorphic determinants and an indirect immunofluorescence technique. Class I antigens were detected in three of the four primary melanomas and in five of the eleven metastatic melanomas. Class II antigens were expressed only in metastatic melanomas, in three out of eleven cases. Some tumour cell suspensions were obtained and short-term cultures were established. Radiobinding and immunoprecipitation studies were carried out in two cases, named M6 and M8. The results were comparable to those obtained with direct immunofluorescence. We modulated the expression of class I and II HLA antigens with interferon in M6 when adapted to tissue culture. This melanoma was class I and II negative; after IFN gamma treatment it became strongly positive for class I and II antigens. In addition we have demonstrated, using Southern blot analysis with the restriction enzymes PvuII and EcoRI, that the M6 melanoma does not have any detectable alterations in its class II beta genes.     

Patterns of major histocompatibility complex class II expression by T cell subsets in different immunological compartments. 2. Altered expression and cell function following activation in vivo

This study characterizes antigen-induced phenotypic and functional aspects of major histocompatibility complex (MHC) class II expression on recirculating T cells in efferent lymph. In vivo secondary, but not primary challenge is associated with both kinetic and phenotypic alterations in class II expression by T cells. All three major T cell subsets, CD4⁺, CD8⁺ and T19⁺ (γδ T cell receptor), show an approximate four fold increase in the level of MHC class II expression during secondary responses. No changes in B cell expression of class II were seen. Resting efferent lymph T cells are predominantly either class II^? or DR⁺DQ^? but this changes to DR⁺DQ⁺ after antigenic challenge. The antigen-presenting function of these class II⁺ T cells was investigated at daily intervals after in vivo antigenic challenge. T cells from non-activated lymph nodes could not induce proliferation of antigen-specific T cells with soluble antigen but were weakly stimulatory in allo-mixed lymphocyte reaction (MLR) at high (> 2:1) stimulator cell ratios. Activated T cells isolated during secondary in vivo responses, and expressing increased quantities of MHC class II, were positive stimulator cells in the MLR. In contrast these cells could not present soluble antigen or trypsin-digested antigen to the T cell lines. In the MLR assays, the relative stimulation by class II⁺ T cells correlates with the levels of class II expression. We conclude from these experiments that both quantitative and qualitative changes in MHC class II, induced on T cells under physiological conditions, play a role in the regulation of the immune response in vivo but that that role is not simply one of presentation of soluble antigen.     

Structure of MHC class I and class II cDNAs and possible immunodeficiency linked to class II expression in the Mexican axolotl

Summary: Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the a chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the lime of hatching. These class 1 genes also present an important polylocism and belong to the same linkage group as the class II B gene, they can be reasonably considered as classical class 1a genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II β chain is relatively well conserved, but most of all does not present any variability in the β domain in inbred as well as in wild axolotls presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class 11 presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lowervertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.     

Topographical expression of class IA and class II phosphoinositide 3-kinase enzymes in normal human tissues is consistent with a role in differentiation

Background  

Growth factor, cytokine and chemokine-induced activation of PI3K enzymes constitutes the start of a complex signalling cascade, which ultimately mediates cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K enzyme family is divided into 3 classes; class I (subdivided into IA and IB), class II (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and class III PI3K. Expression of these enzymes in human tissue has not been clearly defined.     

Peptide trimming by endoplasmic reticulum aminopeptidases: Role of MHC class I binding and ERAP dimerization

Presentation of short peptides, produced through intracellular proteolysis, by MHC class I molecules (MHC-I) is the basis of adaptive immune surveillance and responses by cytolytic CD8⁺ T lymphocytes. In the principal pathway of peptide processing for MHC-I that operates in all nucleated cells, MHC-I-binding peptides are produced through stepwise proteolysis starting with source protein degradation by cytosolic proteasome complexes. Among the fraction of proteasome products reaching the lumen of the endoplasmic reticulum, a significant proportion is thought to have a length exceeding that adapted to MHC class I binding and requires N-terminal trimming. This is carried out by one murine and two human endoplasmic reticulum aminopeptidases, the ERAP enzymes. While the critical role of ERAP for producing a ligandome optimized for MHC-I is well documented, it remains unclear how this is mechanistically achieved. In this review, we will discuss the evidence supporting the alternative “MHC template” and “molecular ruler” models that have been proposed to explain how ERAP activity adapts to the ligand requirements of MHC-I. We will also review evidence for dimerization of the two human ERAP enzymes and its potential functional relevance.     

Asparaginyl endopeptidase: case history of a class II MHC compartment protease

Summary: Although the endpoint of the class II antigen‐processing pathway is well characterized, the processing events that lead to the production of class II major histocompatibility complex (MHC)/peptide complexes are not. It is generally assumed that protease action on native antigen substrates leads to unfolding and capture of either long or short peptides. Whether specific protease activities are needed for presentation of particular T‐cell epitopes is largely unknown. Here, we review our recent studies that aim to identify the processing enzymes that initiate processing of different antigens. We suggest a general strategy that can potentially identify preferred relationships between substrates and processing enzymes in vitro and suggest ways in which these relationships can be tested in vivo. We draw heavily on the example of asparaginyl endopeptidase, which is involved in both productive and destructive processing of different antigen substrates. Overall, while there is undoubtedly redundancy in class II MHC antigen processing, the contributions of individual enzymes can be clearly dissected.     

Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.