Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.     

Strength training and determinants of VO2max in older men

The effects of strength training on maximal aerobic power (VO2max) and some of its determinants were studied in 12 healthy older men (60-72 yr). They underwent 12 wk of strength conditioning of extensors and flexors of each knee with eight repetitions per set, three sets per session, and three sessions per week at 80% of the one repetition maximum (1 RM). Left knee extensors showed a 107% increase in 1 RM, a 10% increase in isokinetic strength at 60 degrees/s, and a 23% increase in total work performed during 25 contractions on an isokinetic dynamometer. Strength measurements of the untrained left elbow extensors showed no change. Leg cycle ergometer VO2max per unit fat-free mass increased by an average 1.9 ml (P = 0.034) whereas arm cycle VO2max was unchanged. Pulmonary function, hemoglobin concentration, erythrocyte volume, plasma volume, and total blood volume did not change. Biopsies of the vastus lateralis showed a 28% increase in mean fiber area, no change in fiber type distribution, a 15% increase in capillaries per fiber, and a 38% increase in citrate synthase activity. The data suggest that the small increase in leg cycle VO2max in older men may be due to adaptations in oxidative capacity and increased mass of the strength-trained muscles.     

The effects of nutrient limitation and stream discharge on the epilithic microbial community in an oligotrophic Arctic stream

Nutrient limitation of epilithic microbial activity is modified by stream discharge and drainage from the tundra surrounding the Kuparuk River, Alaska, USA. During 1984, after three weeks of whole stream enrichment with phosphorus, autotrophic activity per unit biomass had increased in the enriched section of the stream suggesting that phosphorus availability was limiting productivity. In contrast, after three weeks of phosphorus enrichment during 1985, heterotrophic and autotrophic activity was similar in the control and enriched sections of the stream. However, when ammonia or nitrate and phosphorus were added to an in situ bioassay chamber for two weeks, higher community biomass and heterotrophic activity resulted. Ten days later biomass significantly dropped in the unenriched section. Nitrate levels over this period increased four fold concomitantly with decreased stream discharge. Apparently during 1985, nitrogen was limiting epilithic microbial community in the phosphorus enriched section of the Kuparuk River. The significant negative relationship between nitrate concentration and stream discharge observed during 1984 supported the trends seen in 1985. These data suggest that nutrient concentrations which limit epilithic microbial activity and biomass are regulated by the stream discharge and drainage from the surrounding tundra.     

Genetic Studies of Unusual Loci That Affect Body Shape of the Nematode CAENORHABDITIS ELEGANS and May Code for Cuticle Structural Proteins

In Caenorhabditis elegans, four loci (sqt-1, sqt-2, sqt-3 and rol-8) in which mutations affect body shape and cuticle morphology have unusual genetic properties. Mutant alleles of sqt-1 can interact to produce animals with a variety of mutant phenotypes: left roller, right roller, dumpy and long. At least three mutant phenotypes are specified by mutations in the sqt-3 locus. Most alleles at these loci are either dominant or cryptic dominant (i.e., are dominant only in certain genetic backgrounds). Most alleles of these loci exhibit codominance. Two putative null alleles of the sqt-1 locus produce a wild-type phenotype. Many alleles of these genes demonstrate unusual intergenic interactions that are not the result of simple epistasis: animals doubly heterozygous for mutations at two loci often display unexpected and unpredictable phenotypes. We suggest that these genetic properties might be expected of genes, such as the collagen genes, the products of which interact to form the animal's cuticle, and which are member genes of a gene family.     

Does heat shock enhance oxidative stress? Studies with ferrous and ferric iron

Chinese hamster ovary cells were exposed to FeSO4 or FeCl3 during a 43 degrees C heat shock. Concentrations of iron, which were not toxic when cells were incubated at 37 degrees C, became toxic in a dose-dependent fashion during hyperthermia treatment. The iron chelator EDTA, which supports oxidation/reduction reactions, promoted hyperthermia-induced iron cytotoxicity while the iron chelator desferrioxamine, which has been shown to inhibit iron redox cycling, inhibited cytotoxicity. The presence of exogenous superoxide dismutase, catalase, or mannitol during hyperthermia treatment did not inhibit iron toxicity. Depletion of intracellular glutathione by diethylmaleate increased hyperthermia-induced iron toxicity by 76%. These data are interpreted to mean that heat shock promotes intracellular oxidative damage and intracellular glutathione is necessary for protection.     

Peripheral effects of endurance training in young and old subjects

The effects of 12 wk of endurance training at 70% peak O2 consumption (VO2) were studied in 10 elderly (65.1 +/- 2.9 yr) and 10 young (23.6 +/- 1.8 yr) healthy men and women. Training had no effect on weight or body composition in either group. The elderly had more adipose tissue and less muscle mass than the young. Initial peak VO2 was lower in the elderly, but the absolute increase of 5.5-6.0 ml.kg-1.min-1 after training was similar for both groups. Muscle biopsies taken at rest showed that, before training, muscle glycogen stores were 61% higher in the young. Before training, glycogen utilization per joule during submaximal exercise was higher in the elderly. Glycogen stores and muscle O2 consumption increased significantly in response to training in the elderly only. After training, the proportion of energy derived from whole body carbohydrate oxidation during submaximal exercise declined in the young only. The absolute changes that training produced in peak VO2 were similar in both age groups, but the 128% increase in muscle oxidative capacity was greater in the elderly, suggesting that peripheral factors play an important role in the response of the elderly to endurance exercise.     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features.     

Influence of lead exposure on catecholamine metabolism in discrete rat brain nuclei

1. Using the rat exposed both acutely and chronically to lead as a model of lead neurotoxicity, various parameters of catecholamine metabolism were investigated. 2. The steady-state concentrations of noradrenaline, adrenaline and dopamine together with the activities of tyrosine hydroxylase and phenylethanolamine N-methyl transferase were measured in discrete brain nuclei--periventricular, paraventricular, median eminence, posterior and anterior hypothalamus, caudate putamen and globus pallidus. 3. Lead exposure resulted in significant fall in the activity of the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase which was associated with alterations in concentrations of catecholamines in the median eminence, periventricular nucleus and anterior hypothalamus. 4. No other brain nuclei investigated exhibited any effect of lead on the catecholaminergic nervous system and, therefore, the effect of lead on rat brain can be considered to be regionally specific.     

Effects of lead exposure on rat brain catecholaminergic neurochemistry

1. The effects of lead on catecholaminergic neurotransmission have been investigated. 2. Using the rat as a model, animals were exposed both acutely and chronically to lead. The levels of catecholamines, noradrenaline, adrenaline, and dopamine along with the activities of tyrosine hydroxylase and phenylethanolamine-N-methyl transferase were measured in 5 brain regions--cerebral cortex, brainstem, hippocampus, anterior and posterior hypothalamus. 3. A lead related reduction in the activity of tyrosine hydroxylase was observed in association with alterations in steady-state levels of the catecholamines in the posterior and anterior hypothalamus. 4. Thus, lead exposure, known to result in behavioural changes, is associated with localised neurochemical effects on the hypothalamus.