SUMMARY Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in non-neuronal cells are depolymerized by cold, Ca2+ or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability were unclear, studies suggested that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here we show that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have the biochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability in vitro and in vivo. Together, this suggests that transglutaminase-catalyzed polyamination of tubulins stabilizes neuronal microtubules essential for unique neuronal structures and functions.

Background Germline mutations in BRCA1/2 and ATM have been associated with prostate cancer (PCa) risk. Objective To directly assess whether germline mutations in these three genes distinguish lethal from indolent PCa and whether they confer any effect on age at death. Design, setting, and participants A retrospective case-case study of 313 patients who died of PCa and 486 patients with low-risk localized PCa of European, African, and Chinese descent. Germline DNA of each of the 799 patients was sequenced for these three genes. Outcome measurements and statistical analysis Mutation carrier rates and their effect on lethal PCa were analyzed using the Fisher’s exact test and Cox regression analysis, respectively. Results and limitations The combined BRCA1/2 and ATM mutation carrier rate was significantly higher in lethal PCa patients (6.07%) than localized PCa patients (1.44%), p = 0.0007. The rate also differed significantly among lethal PCa patients as a function of age at death (10.00%, 9.08%, 8.33%, 4.94%, and 2.97% in patients who died ≤60 yr, 61–65 yr, 66–70 yr, 71–75 yr, and over 75 yr, respectively, p = 0.046) and time to death after diagnosis (12.26%, 4.76%, and 0.98% in patients who died ≤5 yr, 6–10 yr, and > 10 yr after a PCa diagnosis, respectively, p = 0.0006). Survival analysis in the entire cohort revealed mutation carriers remained an independent predictor of lethal PCa after adjusting for race and age, prostate-specific antigen, and Gleason score at the time of diagnosis (hazard ratio = 2.13, 95% confidence interval: 1.24–3.66, p = 0.004). A limitation of this study is that other DNA repair genes were not analyzed. Conclusions Mutation status of BRCA1/2 and ATM distinguishes risk for lethal and indolent PCa and is associated with earlier age at death and shorter survival time. Patient summary Prostate cancer patients with inherited mutations in BRCA1/2 and ATM are more likely to die of prostate cancer and do so at an earlier age.

SynopsisThe amino-terminal telopeptide of the collagen al(1) chain has a highly conserved sequence. This sequence was analyzed by the Chou-Fasman criteria, and a folded P-sheet conformation, including a P-turn, was predicted. This folded "hairpin" region favors both ionic and hydrophobic intermolecular interactions with al(1) chain residues 930-938 on a neighboring, end-overlapped molecule. An end-overlap interaction of this nature could direct the initial step in fibril formation. The predicted structure also places the potential crosslink-forming lysyl residue, gN, in a unique site a t the /3-turn end of the telopeptide.The type I collagen molecule exists as a long, stiff rod, 15 X 3000 A, composed of two identical al(1) chains and a nonidentical a2 chain. The al(1) chain is 1055 amino acids long; the characteristic triple-helix-forming sequence of repeating -Gly-X-Y-(where X is frequently proline and Y is frequently hydroxyproline) extends over the central 1014 residues.l Short non-triple-helix-forming sequences, called telopeptides,2 comprise the first 16 residues a t the amino terminal of al(1) chain and the last 25 residues at the carboxyl terminal. The telopeptides are rich in tyrosine, which does not occur in the triple-helical region. Each telopeptide contains a lysyl residue which is a potential crosslink precursor after enzymic oxidation to an aldehyde. The telopeptides are susceptible to several proteinases which cannot attack the triple-helical portion of the collagen m~l e c u l e .~-~ Interstitial collagen fibers exhibit a 670-w axial periodicity D, which has been measured both by x-ray diffraction and by electron microscopy. In the generally accepted two-dimensional repre~entation,~ each molecule, -4.40 in length, is displaced axially by 1D from each of its nearest neighbors, leaving an 0.6D gap between axially aligned molecules. The early four-strand6 and five-strand7 microfibril models, as well as more recent analyses,s-10 were designed to preserve the Hodge-Petruska 1D stagger in a three-dimensional ropelike array. The microfibril concept has received verification in small-and medium-angle x-ray diffraction studies of tendons'l and reconstituted collagen fibrils.12

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species (ROS). We surgically induced MI in 6–7 month old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3–4 days after MI or sham surgery (SH). MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca++ sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of CaATP in myofibrillar preparations, which demonstrated a decline in CaATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I (cTnI) and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal nonreducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca++ sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1-hour without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissue. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of <5%. Compared to no fractionation prior to LC-MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four fold. We found approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.

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The proteolytic removal of the extension COOH-terminal propeptide from procollagen has been examined in vitro. A crude enzyme activity was identified in a whole-chicken-embryo extract that acted at acid pH and appeared to be similar to one identified previously [Davidson, J. M., McEneany, L. S. G. & Bornstein, P. (1979) Eur. J. Biochem. 100,[551][552][553][554][555][556][557][558]. This activity was inhibitable by pepstatin but not by leupeptin, suggesting that it might be cathepsin D. Cathepsin D was purified 907-fold from chicken livers by affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and was found to remove the COOH propeptides from procollagen. At pH 6.0, the site of cleavage appeared to shift from the COOH telopeptide to the COOH telopeptide/propeptide junction, based upon the difference in electrophoretic migration of the cleavage products, although determining the actual cleavage site will require end-group analysis. A model for the involvement of cathepsin D in the in vivo processing of procollagen is presented.The enzymes responsible for the sequential removal of the extension propeptides from procollagen, procollagen NH2-terminal proteinase and procollagen COOH-terminal proteinase, have been actively studied, but only the former has been well characterized (1). In vivo it appears that the COOH-terminal propeptide (COOH propeptide) is removed first, with the NH2-terminal propeptide (NH2 propeptide) removed later, possibly after the intermediate has assembled into extracellular fibrils (2-4). The site of COOH propeptide removal has not been well defined. In cell culture experiments, the apparent in vivo pathway is reversed. The COOH propeptide is not cleaved from the product that accumulates in cell culture media, yet conversion is complete in organ culture (5), suggesting that an intact extracellular matrix is required for conversion.In searching for a neutral metalloproteinase that is a COOH-terminal proteinase (6), we observed a cathepsin Dlike activity, which removed the COOH propeptide from procollagen at acid pH. We report here that the cleavage of procollagen with purified cathepsin D is specific for the COOH propeptide and that cleavage occurs at or near the authentic cleavage site when digestion is carried out near pH 6.0. We postulate a role for cathepsin D in the in vivo conversion of procollagen to collagen. MATERIALS AND METHODSProcollagen was isolated from 17-day chicken-embryo tendon fibroblasts (7,8). After tendon dissection and dissociation with collagenase and trypsin, fibroblasts were incubated (2 x 107 cells per ml) for 6 hr in modified Krebs medium II in the presence of 1 ,uCi (1 Ci = 37 GBq) of a mixture of 15 14C-labeled amino acids (New England Nuclear) per ml. Procollagen and partially processed collagen from which the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3302NH2 propeptide was removed (...