A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI,the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis

Background

Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca²⁺.

Methodology

Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme.

Principal Findings

NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI.

Conclusions/Significance

We propose that W542 and Ca²⁺ are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca²⁺ is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.     

Genomewide linkage scan for split-hand/foot malformation with long-bone deficiency in a large Arab family identifies two novel susceptibility loci on chromosomes 1q42.2-q43 and 6q14.1

Split-hand/foot malformation with long-bone deficiency (SHFLD) is a rare, severe limb deformity characterized by tibia aplasia with or without split-hand/split-foot deformity. Identification of genetic susceptibility loci for SHFLD has been unsuccessful because of its rare incidence, variable phenotypic expression and associated anomalies, and uncertain inheritance pattern. SHFLD is usually inherited as an autosomal dominant trait with reduced penetrance, although recessive inheritance has also been postulated. We conducted a genomewide linkage analysis, using a 10K SNP array in a large consanguineous family (UR078) from the United Arab Emirates (UAE) who had disease transmission consistent with an autosomal dominant inheritance pattern. The study identified two novel SHFLD susceptibility loci at 1q42.2-q43 (nonparametric linkage [NPL] 9.8, P=.000065) and 6q14.1 (NPL 7.12, P=.000897). These results were also supported by multipoint parametric linkage analysis. Maximum multipoint LOD scores of 3.20 and 3.78 were detected for genomic locations 1q42.2-43 and 6q14.1, respectively, with the use of an autosomal dominant mode of inheritance with reduced penetrance. Haplotype analysis with informative crossovers enabled mapping of the SHFLD loci to a region of approximately 18.38 cM (8.4 Mb) between single-nucleotide polymorphisms rs1124110 and rs535043 on 1q42.2-q43 and to a region of approximately 1.96 cM (4.1 Mb) between rs623155 and rs1547251 on 6q14.1. The study identified two novel loci for the SHFLD phenotype in this UAE family.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Photo oxidation of rice starch II. Using a water soluble photo initiator

A new photo oxidation system was established when rice starch was oxidized using UV irradiation and 4-(trimethyl ammoniummethyl) benzophenone chloride (BP2) as a photo initiator. BP2 is a water soluble photo initiator. The slurry prepared for photo oxidation contained rice starch, water and BP2 only. No oxidizing agents were added. Parameters affecting the photo oxidation process, i.e. temperature, concentration of BP2, material:liquor ratio and irradiation time were determined. The produced oxidized starch was evaluated by measuring the carboxyl content, carbonyl content and apparent viscosity. The produced photo oxidized rice starch showed sound increase in the carboxyl and carbonyl contents and sharp decrease in the apparent viscosity. The produced photo oxidized starch was tested for its suitability as a sizing agent for cotton yarns. Native starch and oxidized starch, used as a sizing agent by Misr Company of Spinning and Weaving in El-Mahalla El-Kubra (Egypt), were used for comparison. Sized cotton yarns were evaluated by measuring the tensile strength, elongation at break and percent of size removal. Cotton yarns sized using the prepared photo oxidized rice starch showed higher tensile strength, elongation at break and percent of size removal compared with native starch and oxidized starch used by Misr Company of Spinning and Weaving.     

An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Micronization of a Soft Material: Air-Jet and Micro-Ball Milling

The air-jet and ball-mill are frequently used in fine micronization of active pharmaceutical ingredients to the order of 1–5 μm, which is important for increasing dissolution rates, and also for pulmonary delivery. In this study, we investigated the ability of air-jet and ball-mill to achieve adequate micronization on the lab scale using a model soft material, Pluronic® F-68. Material mechanical properties were characterized using the nanometer 600. Pluronic® F-68 was ball-milled in a micro-mill at different material weights and durations in liquid nitrogen vapor. In comparison, a lab scale air-jet mill was used at various milling parameters according to a full factorial design, where the response factors were particle yield and particle size distribution, which was analyzed using laser diffraction and scanning electron microscopy. The yield achieved with the micro-ball mill was 100% but was ~80% for the air-jet mill, which reduced the size of Pluronic® F-68 from 70 μm to sizes ranging between 23–39 μm median diameters. Ball milling produced particles less than 10 μm after 15 min. Although air-jet milling proved capable of particle size reduction of the relatively soft material Pluronic® F-68, limitations to the lower size range achievable were observed. The feed rate of the material into the air jet mill was a significant factor and slower feed rates lead to smaller sizes by allowing more time for particle collisions and subsequent particle breakage to occur. Micro-ball milling under cold condition was more successful at achieving a lower range particle size reduction of soft materials.