Analysis of the topology of ubiquitin chains.

The small protein ubiquitin and its multiple polymers are encountered free in cells and as post-translational modifications on all proteins. Different polyubiquitin three dimensional structures are shown to correlate uniquely with different cellular functions as part of the diverse ubiquitin signaling. At the same time, this multiplicity of structures provides serious challenges to the analytical biochemist. Globally applicable strategies are presented here for the analyses of polyubiquitins and of ubiquitinated proteins, which take advantage of the speed, specificity and sensitivity of top-down tandem mass spectrometry. Particular attention is given to the supervised interpretation of fragmentation as revealed in the MS/MS spectra of these branched proteins. The strategy is compatible with any MS activation technology, is applicable to all polyubiquitin linkage and chain types, can be extended to ubiquitin-like proteins, and will be compatible with and enhanced by continuing advances in LC-MS/MS instrumentation and interpretation software.

Does doxorubicin survive thermal ablation? Results of an ex vivo bench top study.

PURPOSE: We aimed to test the hypothesis that doxorubicin (DOX) survives thermal ablative heating in an ex vivo model of combined transarterial chemoembolization (TACE) and thermal ablation. METHODS: Fresh porcine psoas major muscle (3 samples, 15×10×3 cm) was submerged in aqueous DOX solution (60 µg/mL, 0.1 M) for 24 hours to passively saturate tissue. DOX-infused tissue was then dried and treated with microwave ablation (MWA) using a 2.45 GHz antenna at 65 W for 2, 5, and 10 minutes. Ablations were repeated in triplicate (9 total). Tissue was then sampled at both ablated and unablated control sites, and DOX concentration was quantified via ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), with samples analyzed in triplicate. Tissue DOX levels in ablation and control groups were compared using one-way ANOVA. RESULTS: Homogeneous DOX uptake into porcine tissue was evident in all three samples. Mean DOX concentration in unablated tissue was 8.0±2.2 µg/mL. MWA was technically successful in all 9 procedures (100%), with tissue heating to 95-100°C. Mean tissue DOX concentration showed progressive reduction with increasing ablation time, measuring 6.7±1.3, 4.9±0.9, and 4.8±1.3 µg/mL in MWA-treated tissue after 2, 5, and 10 minutes, respectively. Differences in tissue DOX levels between unablated tissue and MWA groups were statistically significant (P < 0.001). CONCLUSION: Contrary to the initial hypothesis, tissue DOX concentration progressively decreased after MWA of longer ablation times. These results suggest that TACE followed by ablation may result in lower intratumoral DOX than would otherwise be anticipated for TACE alone.

Extrinsic allospecific signals of hematopoietic origin dictate iNKT cell lineage-fate decisions during development.

Invariant NKT (iNKT) cells are critical to the maintenance of tolerance toward alloantigens encountered during postnatal life pointing to the existence of a process for self-education. However, the impact of developmentally encountered alloantigens in shaping the phenotype and function of iNKT cells has not been described. To better understand this process, the current report examined naïve iNKT cells as they matured in an allogeneic environment. Following the prenatal transfer of fetal hematopoietic cells between age-matched allogeneic murine fetuses, cell-extrinsic signals appeared to dictate allospecific patterns of Ly49 receptor expression and lineage diversity in developing iNKT cells. Regulation for this process arose from cells of hematopoietic origin requiring only rare exposure to facilitate broad changes in developing iNKT cells. These findings highlight surprisingly asymmetric allospecific alterations in iNKT cells as they develop and mature in an allogeneic environment and establish a new paradigm for study of the self-education of iNKT cells.

Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry.

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25,600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top-down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation.

Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.

Prenatal Allogeneic Tolerance in Mice Remains Stable Despite Potent Viral Immune Activation.

Transplanting stem cells before birth offers an unparalleled opportunity to initiate corrective treatment for numerous childhood diseases with minimal or no host conditioning. Although long-term engraftment has been demonstrated following in utero hematopoietic cellular transplantation during immune quiescence, it is unclear if prenatal tolerance becomes unstable with immune activation such as during a viral syndrome. Using a murine model of in utero hematopoietic cellular transplantation, the impact of an infection with lymphocytic choriomeningitis virus on prenatal allospecific tolerance was examined. The findings in this report illustrate that established mechanisms of donor-specific tolerance are strained during potent immune activation. Specifically, a transient reversal in the anergy of alloreactive lymphocytes is seen in parallel with the global immune response toward the virus. However, these changes return to baseline following resolution of the infection. Importantly, prenatal engraftment remains stable during and after immune activation. Collectively, these findings illustrate the robust nature of allospecific tolerance in prenatal mixed chimerism compared with models of postnatal chimerism and provides additional support for the prenatal approach to the treatment of congenital benign cellular disease.

Unexpected trypsin cleavage at ubiquitinated lysines.

Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers-linear Ub-(48)Ub-(48)Ub, linear Ub-(63)Ub-(63)Ub, and the branched trimer [Ub]2-(6,48)Ub-have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin.

Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.

Differential patterns of prenatal ipsilateral and contralateral lung growth in cases of isolated left-sided congenital diaphragmatic hernia.

OBJECTIVE: The aim of this research was to compare the impact of varying degrees of visceral herniation on the growth rates of the contralateral and ipsilateral fetal lungs in cases of isolated left-sided congenital diaphragmatic hernia (CDH). METHODS: Data were retrieved from 58 fetuses with isolated left-sided CDH undergoing magnetic resonance imaging studies at both mid-gestation (20-30 weeks) and late-gestation (>30 weeks) time points. The growth of the right and left lungs (ΔLV-R and ΔLV-L) was calculated. The impact of the degree of visceral herniation on the growth disparity between the right and left lungs was then compared. RESULTS: Measurable growth occurred in both lungs between the mid-gestation and late-gestation time points in each group. The ΔLV-R exhibited a strong correlation with ΔLV-L. However, the right lung grew significantly faster than the left lung (ΔLV-R = 1.36 vs ΔLV-L = 0.17 mL/week, P < 0.001). A higher degree of visceral herniation appeared to decrease the growth rate disparity by progressive limitation of the growth of the right lung. CONCLUSION: The contralateral lung retains the potential to grow faster than the ipsilateral lung during the third trimester. A higher degree of visceral herniation places progressive limitations on contralateral lung growth thereby diminishing the growth rate disparity between the right and left lungs.

NK cell tolerance as the final endorsement of prenatal tolerance after in utero hematopoietic cellular transplantation.

The primary benefits of in utero hematopoietic cellular transplantation (IUHCT) arise from transplanting curative cells prior to the immunologic maturation of the fetus. However, this approach has been routinely successful only in the treatment of congenital immunodeficiency diseases that include an inherent NK cell deficiency despite the existence of normal maternal immunity in either setting. These observations raise the possibility that fetal NK cells function as an early barrier to allogeneic IUHCT. Herein, we summarize the findings of previous studies of prenatal NK cell allospecific tolerance in mice and in humans. Cumulatively, this new information reveals the complexity of the fetal immune response in the setting of rejection or tolerance and illustrates the role for fetal NK cells in the final endorsement of allospecific prenatal tolerance.

Expanded intrathoracic space in fetal cases of isolated congenital diaphragmatic hernia contributes to disparity between percent predicted lung volume and observed to expected total lung volume.

OBJECTIVE: The aim of this study was to determine whether fetal lung volume and visceral herniation are associated with changes in intrathoracic space in congenital diaphragmatic hernia(CDH). METHODS: We retrospectively examined the relationship between magnetic resonance imaging-derived measurements of intrathoracic space [predicted lung volume (PLV)] and residual lung volume or visceral herniation among isolated left-sided CDH fetuses. RESULTS: Data from fetal magnetic resonance imaging studies of 60 isolated left-sided CDH cases were analyzed. The median PLV of the CDH fetuses was found to be much greater than the expected total lung volume (eTLV) of a normal fetus at the same gestational age. Surprisingly, liver herniation and observed TLV(oTLV) were positively correlated with the PLV. Although the PPLV was consistently less than the o/eTLV, both indices were greater in survivors than in non-survivors, whereas no significant difference was seen in the PLV/eTLV ratio in regard to survivorship. CONCLUSION: The intrathoracic domain available for lungs and viscera is expanded in CDH fetuses and positively affected by the lung volume and the presence of liver herniation, leading to the difference in the PPLV and o/eTLV. Future study of intrathoracic space as it relates to the growth of the lung and herniated viscera is needed to better characterize the relationship between these parameters.

Preparing to read the ubiquitin code: a middle-out strategy for characterization of all lysine-linked diubiquitins.

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)-based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all-native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS-based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp-selective acid cleavage, separation by reverse phase high-performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine-linked ubiquitin dimers.