Publications by authors named "Mark Luna-Vargas"

Mitochondria maintain a biochemical environment that cooperates with BH3-only proteins (e.g., BIM) to potentiate BAX activation, the key event to initiate physiological and pharmacological forms of apoptosis.

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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.

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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.

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Within the mitochondrial pathway of apoptosis, VDAC2 controls both the localization and proapoptotic activity of BAK. In this issue, Hosoi et al. (2017.

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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.

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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.

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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.

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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.

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Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β 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.

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Article Synopsis
  • Ubiquitin conjugation and deconjugation are crucial for regulating the activity of enzymes through a balance of ubiquitinating enzymes (E1, E2, E3) and deubiquitinating enzymes (DUBs).
  • Different DUBs, especially from the USP family, are regulated by UBL (ubiquitin-like) domains, which play varied roles in enzymatic activity; for instance, USP14's UBL domain boosts catalysis at the proteasome, while USP4's UBL domain inhibits it.
  • USP7’s multiple UBL domains enhance catalysis rather than inhibit, suggesting that UBL domains have distinct functions across USPs, with their activity influenced by other proteins that can modulate their
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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.

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Article Synopsis
  • High-throughput methods are crucial for producing many soluble recombinant protein variants, aiding in the understanding of protein structures and their complexes.
  • The study presents a series of protein expression vectors that enable efficient ligation-independent cloning and co-expression, which helps in screening optimal expression strategies across different proteins.
  • The researchers specifically focused on the ubiquitin specific protease family, successfully creating multiple expression constructs and isolating soluble proteins, demonstrating the versatility of their approach in both E. coli and insect cells for downstream applications like functional studies and crystallization.
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Article Synopsis
  • USP4 is a deubiquitinating enzyme involved in spliceosome regulation, and its structure includes a typical USP-like fold with a ubiquitin-binding site.
  • A ubiquitin-like (Ubl) domain within USP4 can bind to the catalytic region, inhibiting its activity by competing with the ubiquitin substrate.
  • Other USPs, like USP39, can counteract this inhibition, suggesting a regulatory interplay between different USPs.
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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.

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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.

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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.

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