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Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity., Author summary Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea in more than 100 million people worldwide every year. The bacteria replicate in the reproductive tract by evading innate and adaptive immunity. In the absence of effective vaccines and the rise of antibiotic resistance, understanding the molecular interactions between innate immune cells and N. gonorrhoeae may lead to new strategies to combat bacterial growth and the symptoms of gonorrhoea. It has long been known that the N. gonorrhoeae porin, PorB, promotes bacterial survival but also targets host mitochondria in infections. The mechanism by which PorB traffics form the bacterial outer membrane to host mitochondria remains unclear. Here, we utilized proteomics and super-resolution microscopy to show that N. gonorrhoeae secretes PorB via outer membrane vesicles. These vesicles are taken up by macrophages and deliver PorB to mitochondria. Macrophages treated with N. gonorrhoeae vesicles contained damaged mitochondria and active caspase-3. A caspase inhibitor prevented apoptosis of macrophages treated with N. gonorrhoeae vesicles. This suggests that N. gonorrhoeae secretes membrane vesicles, which are readily detectable in gonorrhoea patients, to target macrophages and to promote infections.

Transcriptional dosimetry is an emergent field of radiobiology aimed at developing robust methods for detecting and quantifying absorbed doses using radiation-induced fluctuations in gene expression. A combination of RNA sequencing, array-based and quantitative PCR transcriptomics in cellular, murine and various ex vivo human models has led to a comprehensive description of a fundamental set of genes with demonstrable dosimetric qualities. However, these are yet to be validated in human tissue due to the scarcity of in situ-irradiated source material. This represents a major hurdle to the continued development of transcriptional dosimetry. In this study, we present a novel evaluation of a previously reported set of dosimetric genes in human tissue exposed to a large therapeutic dose of radiation. To do this, we evaluated the quantitative changes of a set of dosimetric transcripts consisting of FDXR, BAX, BCL2, CDKN1A, DDB2, BBC3, GADD45A, GDF15, MDM2, SERPINE1, TNFRSF10B, PLK3, SESN2 and VWCE in guided pre- and post-radiation (2 weeks) prostate cancer biopsies from seven patients. We confirmed the prolonged dose-responsivity of most of these transcripts in in situ-irradiated tissue. BCL2, GDF15, and to some extent TNFRSF10B, were markedly unreliable single markers of radiation exposure. Nevertheless, as a full set, these genes reliably segregated non-irradiated and irradiated tissues and predicted radiation absorption on a patient-specific basis. We also confirmed changes in the translated protein product for a small subset of these dosimeters. This study provides the first confirmatory evidence of an existing dosimetric gene set in less-accessible tissues—ensuring peripheral responses reflect tissue-specific effects. Further work will be required to determine if these changes are conserved in different tissue types, post-radiation times and doses.

Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNP M as a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNP M and redirects it to a step in the viral life cycle. We propose that cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate infection.

SummaryGranzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling.

Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host-pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host-pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host.

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s−1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies — including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry — we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.

Background Sri Lankan Russell’s viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell’s vipers. This study aimed to investigate evidence of myotoxicity in Russell’s viper envenoming, response to antivenom and the toxins responsible for myotoxicity. Methodology and Findings Clinical features of myotoxicity were assessed in authenticated Russell’s viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman’s correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 μg/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 μM, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity. Conclusion The study shows that myotoxicity in Sri Lankan Russell’s viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties., Author Summary There are many gaps in our knowledge of muscle damage caused by snake venoms. Russell’s vipers are more medically important than any other snake in Asia. Sri Lankan Russell’s viper (Daboia russelii) bites have been reported to cause muscle damage in humans, which is not reported for other Russell’s vipers. The aim of the present study was to investigate the onset, severity and resolution of the muscle damage and to identify the toxins responsible for myotoxicity. For this, we studied muscle damage in 245 patients with confirmed Sri Lankan Russell’s viper bites. Patients reported local muscle pain in 72% of cases and generalised muscle pain in 15%. None had severe muscle damage and the symptoms resolved in 80% of patients within 4 days. Measurement of biomarkers of muscle damage in patient blood was consistent with only mild muscle injury, even in patients with symptoms. Two toxins were isolated from Sri Lankan Russell’s viper venom that had similar myotoxic activity to whole venom in chick muscle preparations. Both toxins were weak myotoxins, consistent with what was seen in patients.

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease.

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease.

Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.

Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury; however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury. These experiments show that the disulfide-driven process of nucleocytoplasmic coagulation (NCC) is the main form of injury-induced protein aggregation. NCC is mechanistically distinct from amyloidogenesis, but still broadly impairs cell function by promoting the aggregation of hundreds of abundant and essential intracellular proteins. A small proportion of the intracellular proteome resists NCC and is instead released from necrotic cells. Notably, the physicochemical properties of NCC-resistant proteins are contrary to those of NCC-sensitive proteins. These observations challenge the dogma that liberation of constituents during necrosis is anarchic. Rather, inherent physicochemical features including cysteine content, hydrophobicity and intrinsic disorder determine whether a protein is released from necrotic cells. Furthermore, as half of the identified NCC-resistant proteins are known autoantigens, we propose that physicochemical properties that control NCC also affect immune tolerance and other host responses important for the restoration of homeostasis after necrotic injury.

Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors., Ubiquitin (Ub) receptors are responsible for the recognition of ubiquitylated proteins. Here the authors describe the crystal structure of the ubiquitylated form of the Ub-receptor Rpn10, which suggest that ubiquitylation of Rpn10 promotes its dissociation from the proteasome.

Bacterial proteomic studies frequently use strains cultured in synthetic liquid media over many generations. It is uncertain whether bacterial proteins expressed under these conditions will be the same as the repertoire found in natural environments, or when bacteria are infecting a host organism. Thus, genomic and proteomic characterization of bacteria derived from the host environment in comparison to reference strains grown in the lab, should aid understanding of pathogenesis. Isolates of Corynebacterium pseudotuberculosis were obtained from the lymph nodes of three naturally infected sheep and compared to a laboratory reference strain using bottom-up proteomics, after whole genome sequencing of each of the field isolates. These comparisons were performed following growth in liquid media that allowed us to reach the required protein amount for proteomic analysis. Over 1350 proteins were identified in the isolated strains, from which unique proteome features were revealed. Several of the identified proteins demonstrated a significant abundance difference in the field isolates compared to the reference strain even though there were no obvious differences in the DNA sequence of the corresponding gene or in nearby non-coding DNA. Higher abundance in the field isolates was observed for proteins related to hypoxia and nutrient deficiency responses as well as to thiopeptide biosynthesis.

Nuclear targeting of intact proteasome particles was tested in the Xenopus egg extract system. Both the 26S proteasome holoenzyme and the 20S core particle were targeted to the nuclear envelope but could not enter the nucleus. A novel proteolytically active 20S+ particle was actively imported into the nucleoplasm in a Ran-independent fashion., The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated “+” factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine 48-linked ubiquitin is essential for marking targets for proteasomal degradation, but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine 48 and lysine 63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine 48 linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes., Defective proteasome 19S regulatory particles (RPs) were identified in rpn11f–m1, a proteasomal mutant with mitochondrial phenotypes. The Rpn11 subunit initiates assembly of a five-subunit lid module competent to integrate into pre-assembled base-20S core particle (CP), with subsequent recruitment of remaining lid subunits.

The failure of matrix metalloproteinase (MMP) inhibitor drug clinical trials in cancer was partly due to the inadvertent inhibition of MMP antitargets that counterbalanced the benefits of MMP target inhibition. We explore how MMP inhibitor drugs might be developed to achieve potent selectivity for validated MMP targets yet therapeutically spare MMP antitargets that are critical in host protection.

The matrix metalloproteinases (MMPs) mediate homeostasis of the extracellular environment. They have multiple signalling activities that are commonly altered during tumorigenesis and that might serve as intervention points for anticancer drugs. However, there are many criteria to consider in validating MMPs as drug targets and for the development of MMP inhibitors. The inhibition of some MMPs could have pro-tumorigenic effects (making them anti-targets), counterbalancing the benefits of target inhibition. These effects might partially account for the failure of MMP inhibitors in clinical trials. What are the major challenges in MMP target validation and MMP-inhibitor-drug development?

Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly 2 orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the autoinhibitory role of its C terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared with homogeneous Lys(48)-linked chains. The reduced ability to disassemble homogeneous Lys(48)-linked chains longer than 4 Ub units may prolong residency time on the proteasome.

Zinc-dependent enzymes play important roles in many cellular processes. Assignment of their reaction mechanisms is often a subject of debate because the zinc ion is silent in several spectroscopic techniques. We have combined time-resolved X-ray absorption spectroscopy, pre-steady state kinetics and computational quantum chemistry to study the active site zinc ion of bacterial alcohol dehydrogenase during single substrate turnover. We detect a series of alternations in the coordination number and structure of the catalytic zinc ion with concomitant changes in metal-ligand bond distances. These structural changes are reflected in the effective charge of the metal ion. The present work emphasizes the flexibility of catalytic zinc sites during catalysis and provides novel mechanistic insights into alcohol dehydrogenase catalysis.

The application of x-ray absorption spectroscopy methods to both materials and life sciences is well appreciated. However, the power of extended x-ray absorption fine structure (EXAFS) spectroscopy as a quantitative structural technique has largely been limited by its application to the microscopically homogeneous systems, in which the local environment around each absorbing atom in the sample is the same. The growing interest in time-resolved EXAFS studies of systems in physics, chemistry, biology, and materials science has reintroduced the requirement for an analytical tool to probe heterogeneous mixtures in situ. While long being recognized as a premiere technique for this role, EXAFS studies of mixtures have been particularly difficult due to the strong model dependence and correlations between parameters in the fit. To circumvent these drawbacks, we introduce two new techniques in EXAFS analysis: the principal component analysis and the residual phase analysis. Using a test case of a heterogeneous mi...

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function., Author Summary Animals, plants, fungi, and bacteria all use pore-forming proteins of the membrane attack complex-perforin (MACPF) family as lethal, cell-killing weapons. These proteins are able to insert into the plasma membranes of target cells, creating large pores that short circuit the natural separation between the intracellular and extracellular milieu, with catastrophic results. However, the pore-forming proteins must undergo a substantial transformation from soluble precursors to a large barrel-shaped transmembrane complex as they punch their way into cells. Using a combination of X-ray crystallography and cryo electron microscopy, we have visualized, for the first time, the mechanism of action of one of these pore-forming proteins—pleurotolysin, a MACPF protein from the edible oyster mushroom. This enabled us to propose a model of the pleurotolysin pore by fitting the crystallographic structures of the pore proteins into a three-dimensional map of the pore obtained by cryo electron microscopy. We then designed a set of double mutants that allowed us to chemically trap intermediate states along the trajectory of the pore formation process, and to determine their structures too. By combining these data we proposed a detailed molecular mechanism for pore formation. The pleurotolysin first assembles into rings of 13 subunits, each of which then opens up by about 70° during pore formation. This process is accompanied by refolding and extrusion of two compact regions from each subunit into long hairpins that then zipper together to form an 80-Å wide barrel-shaped channel through the membrane., A combination of structural methods reveals the complex process by which the perforin-like fungal toxin Pleurotolysin rearranges its structure to form a pore that punches a hole in target cell membranes.

International audience; The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neu-rotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate–binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosyn-thesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3–C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC–MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK – an endogenous cytoprotective cascade.

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications., The German Academic Exchange Service (DAAD) and the Michael Smith Foundation for Health Research (MSFHR) supported P.F.H. P.F.L. was supported by the Alexander von Humboldt Foundation, Breast Cancer Society of Canada and MSFHR; L.D.R. was supported by the Canadian Institutes for Health Research (CIHR) and MSFHR; and U.E. was supported by the MSFHR. This work was supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology (C.M.O.), a grant from the CIHR (grant number M.O.P. 111055) as well as by an Infrastructure Grant from MSFHR and the Canada Foundations for Innovation (C.M.O.). Further support was provided by the European Union FP7 program; Consolider Program of the Spanish Ministry of Science and Technology; and State Plan for Research in Science, Technology and Innovation of the Spanish Ministry of Economy and Competitiveness (F.X.G.-R.)

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases (1). It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.

International audience; In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mito-chondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent sub-strate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S protea-some activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging.

Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis.

EMBO J 28, 1867–1877 (2009); published online 08 July 2009 [PMC free article] [PubMed] Polyubiquitin (polyUb) is a diverse signal in terms of both chain length and linkage type (Ikeda and Dikic, 2008). Chains can be formed through covalent conjugation of ubiquitin (Ub) to any of the seven lysine residues on the preceding Ub (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, or Lys63), and in some instances two Ub molecules can simultaneously modify two lysine residues on a single Ub resulting in branched chains (Kim et al, 2007). Ubiquitin chain-length and linkage type, along with affinity for proteasome receptors and ease of deubiquitination, all contribute towards setting substrate hierarchy. Hence, polyUb-binding proteins influence the specificity and efficiency of intra-cellular proteolysis. One such auxiliary factor, S5a (or Rpn10), partakes in shuttling polyUb conjugates, limiting the access of competing substrate carriers, and in anchoring Ub chains at the proteasome (Deveraux et al, 1994; Glickman et al, 1998; Matiuhin et al, 2008). It now seems that S5a/Rpn10 also functions in upstream events by blocking the synthesis of low-priority forked chains and promoting the formation of unbranched chains with high affinity for the proteasome (Kim et al, 2009). In an attempt to understand why some conjugation reactions generate polyubiquitinated substrates that are poorly degraded, and how Ub-binding proteins support proteosomal degradation, Kim et al have added purified S5a to a coupled ubiquitination–degradation assay. The presence of S5a in the reaction containing a substrate (luciferase), ubiquitination enzymes (an E2, UbcH5, and the U-box E3 ligase CHIP) and 26S proteasome significantly increased the rate of luciferase degradation. S5a had a similar effect on the degradation of another substrate (troponin I) ubiquitinated by UbcH5 and another E3 ubiquitin ligase (MuRF1). The yeast homologue of S5a, Rpn10, enhanced the proteasomal degradation of troponin I to the same extent as S5a. In order to understand the source of proteolysis enhancement, S5a was then added to the reaction mix after the formation of ubiquitin conjugates. This move actually inhibited the degradation of the luciferase substrate, indicating that S5a is involved in early steps of targeting proteins for degradation. The use of mutated ubiquitin restricted in the linkages it can form (K48R, K11R, K29R, or K63R) did not alter the effect of S5a on degradation, indicating that degradation is not achieved by increasing the levels of any specific chain linkage. Nevertheless, mass spectrometry analysis of conjugates formed by CHIP with UbcH5 showed a decrease in forked linkages when S5a was added to the ubiquitination reaction. Forked chains are those in which two Ub molecules are linked to two adjacent lysines on the preceding Ub molecule (in essence, rather than forming elongated chains, some polyUbs might exist as a branched bush; see Figure 1A). It is important to note that because of technical limitations only simultaneous modification on adjacent lysines (K6/11, K27/29, or 29/33) was assayed in the accompanying study. The effect of S5a on the formation of forked chains was also detected during ubiquitination by the MuRF1 and UbcH5 pair of enzymes. Hydrolysis of substrates linked to homogenous non-forked Ub chains was not enhanced by addition of S5a and the authors therefore conclude that S5a interacts with the growing polyUb chain to prevent fork formation. Figure 1 The ubiquitin-binding protein, S5a/Rpn10, trails ubiquitin conjugates along their trajectory. (A) A general scheme of the ubiquitin–proteasome pathway updated with new results described by Kim et al (2009) in this issue. Ubiquitination enzymes ... What hurdle do forked Ub chains pose on protein degradation? Part of the effect of S5a seems to stem from the difficulty in processing (or deubiquitinating) forked chains. In an elegant set of experiments, the authors go on to show that forked Ub chains bind poorly to 26S, in contrast to non-forked ones. In a competition assay, luciferase linked to mixed forked Ub chains (ubiquitinated in the absence of S5a) was unable to inhibit the proteasomal degradation of troponin I linked to Lys63 or Lys48 chains, whereas luciferase linked to Lys63 chains or mixed non-forked chains (ubiquitinated in the presence of S5a) caused a significant decrease in the degradation of troponin I bound to homogeneous chains. Furthermore, in a binding assay, MuRF1 autoubiquitinated in the presence of S5a showed higher affinity for purified 26S than MuRF1 ubiquitinated in the absence of S5a. Poor anchoring to the proteasome (by S5a or another receptor; Figure 1A) might be the cause for the apparent stability and slow rate of processing or proteolysis of these conjugates. The accompanying manuscript by Kim et al (2009) opens up a porthole to an exciting new layer of complexity in directing the ubiquitination process. Ubiquitin chain length and linkage type, along with the affinity for proteasome receptors and ease of deubiquitination, all contribute towards setting substrate hierarchy (Figure 1A). To these selection processes, one can now add a new checkpoint: at early stages of ubiquitination S5a limits the formation of forked chains, in which chains are extended at more than one lysine on a given Ub. Yet, as with many new observations, some amount of caution should be exercised when considering the general implications. So far branched chains have been identified mostly in vitro and their formation is strongly depended on the E2 used in the ubiquitination reaction. Among all possible branched modifications, only three forks at adjacent lysines on a single ubiquitin (K6/11, K27/29, and K29/33) have been documented. An accurate quantification of branched ubiquitin relative to total Ub-in-chains is yet to be carried out, although the assumption is that the ratio of forked modifications over extended chains is low (Kim et al, 2009). Furthermore, S5a UIM binds Ub through a patch of hydrophobic residues (Walters et al, 2002), leaving most lysine residues exposed for unrestricted conjugation to subsequent Ub (Figure 1B). This implies an intricate mechanism for S5a to block synthesis of forked chains, involving interactions between multiple components of the synthesis machinery, perhaps by getting polyubiquitinated itself, thus deflecting the imprecise ubiquitination of the substrate (Kim et al, 2009). Nonconformity in ubiquitin polymerization—e.g. arborization in contrast to extension—might lead to accumulation of highly stable ubiquitin conjugates that are poorly recognized and slow to be removed. As previously shown in vivo, expression of Rpn10 or its UIM domain alone was able to reverse the accumulation of poorly turned-over polyUb conjugates under an induced stress condition (Matiuhin et al, 2008). The corrective properties of S5a in directing proper chain formation and prioritizing proteasome-bound substrates might be useful for enhancing protein turnover or overcoming stress conditions associated with malfunctions in the ubiquitin–proteasome system.

Previous studies utilizing apolipoprotein E (apoE)-deficient mice revealed distinct decreases in the levels of cholinergic synaptic markers of projecting basal forebrain cholinergic neurons and no such alterations in other brain cholinergic systems. In order to investigate the mechanisms underlying these neuron-specific cholinergic effects, primary neuronal cultures from apoE-deficient and control mice were prepared and characterized. These include basal forebrain cultures, which are enriched in projecting cholinergic neurons, and cortical cultures, which contain cholinergic interneurons. The levels of cholinergic nerve terminals in these cultures were assessed by ligand binding measurements of the levels of the vesicular acetylcholine transporter (VAChT). This revealed that basal forebrain cultures of apoE-deficient mice contain markedly lower VAChT levels (approximately 50%) than do control cultures, but that VAChT levels of the corresponding cortical cultures of the apoE-deficient and control mice were the same. Time course studies revealed that VAChT levels of the basal forebrain cultures increased with culture age, but that the relative reduction in VAChT levels of the apoE-deficient cholinergic neurons was unaltered and was the same for freshly prepared and for 96 h old cultures. These in vitro observations are in accordance with the in vivo findings and suggest that projecting basal forebrain cholinergic neurons, but not cholinergic interneurons, are markedly dependent on apoE and that similar mechanisms mediate the in vivo and in vitro effects of apoE deficiency on cholinergic function.

Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin.

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo–N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible α- and ɛ-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity.

International audience; Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.

Functional redundancy of Rpn5 in budding yeast allows its participation and function in two distinct but structurally related PCI complexes: the proteasome lid and the CSN. As a lid subunit, Rpn5 stabilizes proteasome integrity; as a CSN subunit, Rpn5 is required for enzymatic hydrolysis of Rub1/Nedd8 from cullins., Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

Determining the sequence of protein N-termini and their modifications functionally annotates proteins since translation isoforms, posttranslational modifications, and proteolytic truncations direct localization, activity, and the half-life of most proteins. Here we present in detail the steps required to perform our recently described approach we call Terminal Amine Isotopic Labeling of Substrates (TAILS), a combined N-terminomics and protease substrate discovery degradomics platform for the simultaneous quantitative and global analysis of the N-terminome and proteolysis in one MS/MS experiment. By a 3-day procedure with flexible α- and ɛ-amine labeling and blocking options, TAILS removes internal tryptic and C-terminal peptides by binding to a dendritic polyglycerol aldehyde polymer. Therefore, by negative selection, this enriches for both the N-terminal-labeled peptides and all forms of naturally blocked N-terminal peptides. In addition to providing valuable proteome annotation, the simultaneous analysis of the original mature N-terminal peptides enables these peptides to be used for higher confidence protein substrate identification by two or more different and unique peptides. Second, the analysis of the N-terminal peptides forms a statistical classifier to determine valid isotope ratio cutoffs in order to identify with high-confidence protease-generated neo-N-terminal peptides. Third, quantifying the loss of acetylated or cyclized N-terminal peptides that have been cleaved extends overall substrate coverage. Hence, TAILS allows for the global analysis of the N-terminome and determination of cleavage site motifs and substrates for protease including those with unknown or broad specificity.

Summary eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-β Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-β is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physically linked to facilitate efficient protein synthesis.

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP(+)/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.

Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.

The understanding of structure-function relationships in proteins has been significantly advanced with the advent of the biotechnological revolution. A goal yet to be realized for many metalloenzyme systems is to characterize the dynamic changes in structure that bridge the static endpoints provided by crystallography. We present here a series of edge and EXAFS spectra of the metalloenzyme alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) complexed with its substrate. The enzyme-substrate complexes were trapped by fast freezing at various times, following their enzyme activity. Our edge and EXAFS analyses both reveal the time-dependent changes in the structure of the active site of TbADH.

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.