Altered Intersubunit Communication Is the Molecular Basis for Functional Defects of Pathogenic p97 Mutants

The human AAA ATPase p97 is a molecular chaperone essential in cellular proteostasis. Single amino acid substitutions in p97 have been linked to a clinical multiple-disorder condition known as inclusion body myopathy associated with Paget''s disease of the bone and frontotemporal dementia. How the mutations affect the molecular mechanism that governs the function of p97 remains unclear. Here, we show that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of adenosine-5′-O-(3-thiotriphosphate) binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate.     

Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.     

Synthesis and antitumor activity of 1,3,4-oxadiazole possessing 1,4-benzodioxan moiety as a novel class of potent methionine aminopeptidase type II inhibitors

A series of 1,3,4-oxadiazole derivatives containing 1,4-benzodioxan moiety (7a–7q) have been designed, synthesized and evaluated for their antitumor activity. Most of the synthesized compounds were proved to have potent antitumor activity and low toxicity. Among them, compound 7a showed the most potent biological activity against Human Umbilical Vein Endothelial cells, which was comparable to the positive control. The results of apoptosis and flow cytometry (FCM) demonstrated that compound 7a induce cell apoptosis by the inhibition of MetAP2 pathway. Molecular docking was performed to position compound 7a into MetAP2 binding site in order to explore the potential target.     

Synthesis and antitumor-evaluation of 1,3-selenazole-containing 1,3,4-thiadiazole derivatives

A series of novel 1,3-selenazole-containing 1,3,4-thiadiazole derivatives bearing Schiff base moieties were synthesized and evaluated for their in vitro antiproliferative activities against human breast cancer cell MCF-7 and mouse lymphocyte leukemia cell L1210 by CCK-8 assay. The majority of the compounds showed better activity against MCF-7 cell, compared with lead compound PCS. In particular, compound 6c was the most potent compound with IC₅₀ value of 4.02 μM.     

Synthesis,structure analysis,antitumor evaluation and 3D-QSAR studies of 3,6-disubstituted-dihydro-1,2,4,5-tetrazine derivatives

3,6-Diaryl-dihydro-1,2,4,5-tetrazine derivatives were synthesized and their structures were confirmed by single-crystal X-ray diffraction. Monosubstituted dihydrotetrazines are the 1,4-dihydro structure, but disubstituted dihydrotetrazines are the 1,2-dihydro structure. The results of further research indicated there may be a rearrangement during the synthesis process of disubstituted dihydrotetrazines. Their antitumor activities were evaluated against A-549 and P388 cells in vitro. The results showed several compounds to be endowed with cytotoxicity in the low micromolar range. Two compounds were highly effective against A-549 cell and IC₅₀ values were 0.575 and 2.08 μM, respectively. Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies of comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were carried out on 37 1,2,4,5-tetrazine derivatives with antitumor activity against A-549 cell. Models with good predictive abilities were generated with the cross validated q² values for CoMFA and CoMSIA being 0.744 and 0.757, respectively. Conventional r² values were 0.978 and 0.988, respectively, the predicted R² values were 0.916 and 0.898, respectively. The results provide the tool for guiding the design and synthesis of novel and more potent tetrazine derivatives.     

Multidimensional optimization of promising antitumor xanthone derivatives

A promising antitumor xanthone derivative was optimized following a multidimensional approach that involved the synthesis of 17 analogues, the study of their lipophilicity and solubility, and the evaluation of their growth inhibitory activity on four human tumor cell lines. A new synthetic route for the hit xanthone derivative was also developed and applied for the synthesis of its analogues. Among the used cell lines, the HL-60 showed to be in general more sensitive to the compounds tested, with the most potent compound having a GI₅₀ of 5.1 μM, lower than the hit compound. Lipophilicity was evaluated by the partition coefficient (K_p) of a solute between buffer and two membrane models, namely liposomes and micelles. The compounds showed a log K_p between 3 and 5 and the two membrane models showed a good correlation (r² = 0.916) between each other. Studies concerning relationship between solubility and structure were developed for the hit compound and 5 of its analogues.     

Activity rhythms of enzymes in human red blood cell suspensions

Abstract

We have established the presence of a rhythm in the activity of 4 enzymes in in‐vitro cell suspensions of human red blood cells. Glucose 6‐phosphate dehydrogenase and glutamate oxaloacetate transaminase demonstrated semicircadian patterns of activity, while acid phosphatese and acetylcholine esterase exhibited circadian activity rhythms. The ratios between the highest to lowest activities varied from 2:1 to 10:1 among the various enzymes. The affinity of glucose 6 phosphate dehydrogenase to its substrate and coenzyme remained constant throughout the cycle. No evidence was obtained for the presence of a soluble inhibitor at the lower levels of the activity. Sonication of hemolysates with low glucose 6 phosphate dehydrogense activity yielded additional activity comparable to that of the peak activity. Sonication of hemolysates from the time of the peak activity did not change the original activity. The observations point to a role of the cell membrane in the biological clock.     

Phase control of circadian activity in the antelope ground squirrel

Abstract

Wheel‐running activity of forty antelope ground squirrels, Ammospermophilus leucurus, was monitored for several months in both an outdoor cage and in the laboratory. The squirrels demonstrated a highly diurnal pattern which persisted in “constant conditions.” After removal from the field the initial free‐running period was close to 24 hrs, but typically lengthened in a nearly linear fashion at least for the first few months. There was no evidence of any difference in this trend for squirrels, in D/D, L/L 100 lx, 250 lx or 1200 lx. Eventually, about 90 percent of the squirrels had periods longer than 24 hrs.

The synchronizing capacity of the natural photoperiod was used to “catch the free‐running rhythm” and thereby demonstrate a response curve. Synchronization occurred by a shortening of the period when the time of sunrise was between 125° and 0° (subjective night) and by a lengthening of the period when the time of sunrise was between 0° and 125° (subjective day).

To more thoroughly examine the underlying mechanisms of phase control, phase‐response curves based on sixty one light‐pulse experiments were constructed. Comparisons of curves based on 6‐hr and 15‐min pulses, showed that the integral action of light is important (i.e., the entire pulse is involved in phase shifting). It was found that light pulses not only affected the phase of the rhythm but also the phase. Large phase shifts were usually associated with decreases in free‐running period. Several hypotheses on the controlling mechanisms were advanced.     

A change in the pattern in circadian running‐wheel activity after lesions of the intergeniculate leaflet and ventral lateral geniculate nucleus in the Syrian hamster

Abstract

The suprachiasmatic nuclei (SCN) contain the endogenous mammalian circadian pacemaker, which generates the circadian rhythm in locomotor activity. In Syrian hamsters with free‐running rhythms, the onset of running‐wheel activity is very precise and predictable while the end (offset) is more variable. From the thalamic intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (vLGN) a projection to the SCN originates. Animals with a lesion aimed at the IGL/vLGN and sham‐and unoperated controls were kept in continuous darkness. With linear regression, lines were fitted through 10 successive onsets and offsets of activity and the mean deviation of the onsets and offsets from the fitted lines was determined. Animals with a complete or partial lesion of the IGL/vLGN had a smaller mean deviation of the circadian activity offset from the fitted regression line (0.313 h) compared with the grouped control animals (0.678 h). To test the difference statistically, we compared the sum of the square residuals of the circadian offsets between the groups. This difference was highly significant (F(69,64)=4.16, p<0.0001), which indicates that animals with a lesion of the IGL/ vLGN have a less variable circadian offset of running‐wheel activity. No differences were observed in the variability in the circadian onset of locomotor activity between experimental and control animals. It is concluded that the IGL/vLGN influence the variability of the offset of the circadian running‐wheel activity.