MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain.

USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.

Randomized controlled trial of a group peer mentoring model for U.S. academic medicine research faculty.

Introduction: Midcareer is a critical transition point for biomedical research faculty and a common dropout point from an NIH-funded career. We report a study to assess the efficacy of a group peer mentoring program for diverse biomedical researchers in academic medicine, seeking to improve vitality, career advancement, and cross-cultural competence. Methods: We conducted a stratified randomized controlled trial with a waitlist control group involving 40 purposefully diverse early midcareer research faculty from 16 states who had a first-time NIH R01 (or equivalent) award, a K training grant, or a similar major grant. The yearlong intervention (2 to 3 days quarterly) consisted of facilitated, structured, group peer mentoring. Main study aims were to enhance faculty vitality, self-efficacy in achieving research success, career advancement, mentoring others, and cultural awareness and appreciation of diversity in the workplace. Results: Compared to the control group, the intervention group's increased vitality did not reach statistical significance (P = 0.20), but perceived change in vitality was 1.47 standard deviations higher (D = 1.47, P = 0.03). Self-efficacy for career advancement was higher in the intervention group (D = 0.41, P = 0.05) as was self-efficacy for research (D = 0.57, P = 0.02). The intervention group also valued diversity higher (D = 0.46, P = 0.02), had higher cognitive empathy (D = 0.85, P = 0.03), higher anti-sexism/racism skills (D = 0.71, P = 0.01), and higher self-efficacy in mentoring others (D = 1.14, P = 0.007). Conclusions: The mentoring intervention resulted in meaningful change in important dimensions and skills among a national sample of diverse early midcareer biomedical faculty. This mentoring program holds promise for addressing the urgencies of sustaining faculty vitality and cross-cultural competence.

The role of UBL domains in ubiquitin-specific proteases.

Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin.

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases.

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.

Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes.

We demonstrate that oxime ligation is an efficient, straightforward, and generally applicable strategy for generating nonhydrolyzable ubiquitin (Ub)-isopeptide isosteres. We synthesized nonhydrolyzable K48- and K63-linked Ub-isopeptide isosteres to investigate the selectivity of deubiquitinating enzymes for specific linkages employing surface plasmon resonance spectroscopy. The results indicate that deubiquitinating enzymes specifically recognize the local peptide sequence flanking Ub-branched lysine residues in target proteins. The described strategy allows the systematic investigation of sequence requirements for substrate selectivity of deubiquitinating enzymes.

Mitochondrial Isolation and Real-Time Monitoring of MOMP.

Isolated model systems have proven to be the standard in the apoptosis field to deconstruct MOMP into individual steps and to study the behavior of a subset of MOMP regulators. Here we describe the method to isolate, JC-1-label, and purify mouse liver mitochondria and subsequently describe how to utilize the JC-1-labeled mitochondria for real-time MOMP measurements.

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins.

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.

The peroxisomes strike BAK: Regulation of peroxisome integrity by the Bcl-2 family.

Within the mitochondrial pathway of apoptosis, VDAC2 controls both the localization and proapoptotic activity of BAK. In this issue, Hosoi et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605002) find that loss of VDAC2 diverts BAK into peroxisome membranes, revealing the ability of BAK to control peroxisome membrane integrity and the release of soluble peroxisomal matrix proteins.

The deadly landscape of pro-apoptotic BCL-2 proteins in the outer mitochondrial membrane.

Apoptosis is a biological process that removes damaged, excess or infected cells through a genetically controlled mechanism. This process plays a crucial role in organismal development, immunity and tissue homeostasis, and alterations in apoptosis contribute to human diseases including cancer and auto-immunity. In the past two decades, significant efforts have focused on understanding the function of the BCL-2 proteins, a complex family of pro-survival and pro-apoptotic α-helical proteins that directly control the mitochondrial pathway of apoptosis. Diverse structural investigations of the BCL-2 family members have broadened our mechanistic understanding of their individual functions. However, an often over-looked aspect of the mitochondrial pathway of apoptosis is how the BCL-2 family specifically interacts with and targets the outer mitochondrial membrane to initiate apoptosis. Structural information on the relationship between the BCL-2 family and the outer mitochondrial membrane is missing; likewise, knowledge of the biophysical mechanisms by which the outer mitochondrial membrane affects and effects apoptosis is lacking. In this mini-review, we provide a current overview of the BCL-2 family members and discuss the latest structural insights into BAK/BAX activation and oligomerization in the context of the outer mitochondrial membrane and mitochondrial biology.

Cell Biology: ERADicating Survival with BOK.

Mechanistic insights into the function of the pro-apoptotic BCL-2 family member BOK have remained elusive. A recent study shows that healthy cells constitutively degrade BOK via the ER-associated degradation and ubiquitin-proteasome pathways; following proteasome inhibition, BOK is stabilized to initiate a unique pro-apoptotic death program.

Physiological and Pharmacological Control of BAK, BAX, and Beyond.

Cellular commitment to the mitochondrial pathway of apoptosis is accomplished when proapoptotic B cell chronic lymphocytic leukemia/lymphoma (BCL)-2 proteins compromise mitochondrial integrity through the process of mitochondrial outer membrane permeabilization (MOMP). For nearly three decades, intensive efforts focused on the identification and interactions of two key proapoptotic BCL-2 proteins: BCL-2 antagonist killer (BAK) and BCL-2-associated X (BAX). Indeed, we now have critical insights into which BCL-2 proteins interact with BAK/BAX to either preserve survival or initiate MOMP. In contrast, while mitochondria are targeted by BAK/BAX, a molecular understanding of how these organelles govern BAK/BAX function remains less clear. Here, we integrate recent mechanistic insights of proapoptotic BCL-2 protein function in the context of mitochondrial environment, and discuss current and potential pharmacological opportunities to control MOMP in disease.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).

The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange.

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-ß response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP-Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP-Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP-Ubl domain promotes a change of a switching loop near the active site. This 'allosteric regulation of product discharge' provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes.

The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types.

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.

Usp39 is essential for mitotic spindle checkpoint integrity and controls mRNA-levels of aurora B.

Accurate chromosome segregation relies on the mitotic spindle checkpoint. This checkpoint acts to restrict ubiquitin ligase activity of the Anaphase-promoting complex (APC/C) in mitosis until all chromosomes are bipolarly attached to the mitotic spindle. We performed a functional RNAi-based screen to identify De-ubiquitinating enzymes (Dubs) involved in mitotic progression. We identified Usp39 as a new factor required to maintain the spindle checkpoint and support successful cytokinesis. Strikingly, although Usp39 clearly contains an ubiquitin-protease domain, we show that Usp39 is entirely deprived of Dub activity. However, consistent wilt a previously described role for Usp39 in mRNA processing, we observed specific reduction in Aurora B-mRNA levels after depletion of Usp39. Although we find that exogenously expressed Aurora B cDNA is not sufficient to rescue the checkpoint defect of Usp39-depleted cells, Aurora B expression is restored. Our observations suggest Usp39 to be involved in splicing of Aurora B and other mRNAs that are essential for proper spindle checkpoint function.

A genomic and functional inventory of deubiquitinating enzymes.

Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

Profiled bar transmission gratings: soft-x-ray calibration of new Kirchoff solutions.

A new analytical model, derived rigorously from scalar diffraction theory, accurately fits soft-x-ray measurements of symmetrical profile gold transmission gratings in all diffracted orders. The calibration system selects numerous photon energies by use of a high-resolution grazing-incidence monochromator and a dc e-beam source. Fine-period free-standing gratings exhibit limited performance and require such testing to determine parameters of and select acceptable gratings for use in time-resolved (0.25 ns) spectrographs of known radiometric response. Unfolded spectra yield a Z-pinch plasma peak kT approximately 250 eV, total radiated energy approximately 900 kJ, and a pinch-driven gold-wall hohlraum Planckian kT approximately 86 eV.