Lrp1/LDL Receptor Play Critical Roles in Mannose 6‐Phosphate‐Independent Lysosomal Enzyme Targeting

Most lysosomal enzymes require mannose 6‐phosphate (M6P) residues for efficient receptor‐mediated lysosomal targeting. Although the lack of M6P residues results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types, suggesting the existence of M6P‐independent transport routes. Here, we quantify the lysosomal proteome in M6P‐deficient mouse fibroblasts (PT^ki) using Stable Isotope Labeling by Amino acids in Cell culture (SILAC)‐based comparative mass spectrometry, and find unchanged amounts of 20% of lysosomal enzymes, including cathepsins D and B (Ctsd and Ctsb). Examination of fibroblasts from a new mouse line lacking both M6P and sortilin, a candidate for M6P‐independent transport of lysosomal enzymes, revealed that sortilin does not act as cargo receptor for Ctsb and Ctsd. Using fibroblast lines deficient for endocytic lipoprotein receptors, we could demonstrate that both LDL receptor and Lrp1 mediate the internalization of non‐phosphorylated Ctsb and Ctsd. Furthermore, the presence of Lrp1 inhibitor increased the secretion of Ctsd from PT^ki cells. These findings establish Lrp1 and LDL receptors in M6P‐independent secretion‐recapture targeting mechanism for lysosomal enzymes.      

Prepared for the future: A strong signal of evolution toward the adult benthic niche during the pelagic stage in Labrid fishes

The morphology of organisms reflects a balance between their evolutionary history, functional demands, and biomechanical constraints imposed by the immediate environment. In many fish species, a marked shift in the selection regime is evident when pelagic larvae, which swim and feed in the open ocean, settle in their adult benthic habitat. This shift is particularly dramatic in coral‐reef fishes, where the adult habitat is immensely complex. However, whether the adult trophic ecotype affects the morphology of early‐life stages is unclear. We measured a suite of 26 functional‐morphological traits in the head and body of larvae from an ontogenetic series of 16 labrid species. Using phylogenetic comparative methods, we reconstructed the location of adaptive peaks of larvae whose adults are associated with different trophic ecotypes. We found that the morphospace occupation in these larvae is largely driven by divergent adaptations to the adult benthic habitats. The disparity between adaptive peaks is achieved early and does not monotonically increase with size. Our findings thus refute the notion that larvae rapidly acquire the trophic‐specific traits during a metamorphic period immediately prior to settlement. This early specialization might be due to the highly complex musculoskeletal system of the head that cannot be rapidly modified.     

Oligo(dT) is not a correct native PAGE marker for single-stranded DNA

Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility, and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length, and gel concentration. Depending on their length, oligo(dT) fragments migrate 2--4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g., determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.     

Surfactant protein C and lung function: new insights into the role of α-helical length and palmitoylation

Surfactant protein C (SP-C) is known to be essential for lung function and the formation of a surface confined reservoir at the alveolar interface. The structural features relevant for the peptide’s extraordinary ability to form extended three-dimensional structures were systematically investigated and are summarized in the present paper. The influence of palmitoylation was studied for full length SP-Cs as well as truncated variants with the N-terminal residues 1–17 and 1–13, respectively. The combined results from film balance measurements, fluorescence microscopy (FLM) and scanning force microscopy (SFM) reveal a fine-tuned balance between the influence of the palmitoyl chains and α-helical length. Native SP-C added to DPPC/DPPG monolayers (molar ratio 80:20) induced the formation of the surface confined reservoir independent of its palmitoylation degree. However, topographic images revealed that only bilayers and not multilayers where formed when the acyl chains were missing. The influence of palmitoylation increased when α-helical length was considerably reduced to 17 or even 13 amino acid residues. In these strongly truncated SP-C peptides palmitoyl chains increased monolayer stability and anchored the peptides in the lipid film. However, no multilayer formation was observed at all for all shortened peptides. The α-helix of SP-C seems to be a prerequisite for the formation of extended three-dimensional structures and obviously has to be able to span a lipid bilayer. Palmitoylation obviously mediates interactions between lipids and/or peptides not only within a protein/lipid film but also between neighbouring layers and induces a stacking of bilayers. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing

The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing.     

Rapid conditional knock-down-knock-in system for mammalian cells

RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down-knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.     

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition

Purpose To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. Experimental design Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. Results Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors’ monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P < 0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68− CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. Conclusions Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.     

IMCL area density, but not IMCL utilization, is higher in women during moderate-intensity endurance exercise, compared with men

Women use more fat during endurance exercise as evidenced by a lower respiratory exchange ratio (RER). The contribution of intramyocellular lipid (IMCL) to lipid oxidation during endurance exercise is controversial, and studies investigating sex differences in IMCL utilization have found conflicting results. We determined the effect of sex on net IMCL use during an endurance exercise bout using an ultrastructural evaluation. Men (n = 17) and women (n = 19) completed 90-min cycling at 63% Vo(2peak). Biopsies were taken before and after exercise and fixed for electron microscopy to determine IMCL size, # IMCL/area, IMCL area density, and the % IMCL touching mitochondria. Women had a lower RER and carbohydrate oxidation rate and a higher lipid oxidation rate during exercise (P < 0.05), compared with men. Women had a higher # IMCL/area and IMCL area density (P < 0.05), compared with men. Women, but not men, had a higher % IMCL touching mitochondria postexercise (P = 0.03). Exercise decreased IMCL area density (P = 0.01), due to a decrease in the # IMCL/area (P = 0.02). There was no sex difference in IMCL size or net use. In conclusion, women have higher IMCL area density compared with men, due to an increased # IMCL and not an increased IMCL size, as well as an increased % IMCL touching mitochondria postexercise. Endurance exercise resulted in a net decrease in IMCL density due to decreased number of IMCL, not decreased IMCL size, in both sexes.     

Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.