Neocarzilin Inhibits Cancer Cell Proliferation via BST-2 Degradation, Resulting in Lipid Raft-Trapped EGFR.

Neocarzilin (NCA) is a natural product exhibiting potent antimigratory as well as antiproliferative effects. While vesicle amine transport protein 1 (VAT-1) was previously shown to inhibit migration upon NCA binding, the molecular mechanisms responsible for impaired proliferation remained elusive. We here introduce a chemical probe closely resembling the structural and stereochemical features of NCA and unravel bone marrow stromal antigen 2 (BST-2) as one of the targets responsible for the antiproliferative effect of NCA in cancer cells. The antiproliferative mechanism of NCA was confirmed in corresponding BST-2 knockout (KO) HeLa cells, which were less sensitive to compound treatment. Vice versa, reconstitution of BST-2 in the KO cells again reduced proliferation upon NCA addition, comparable to that of wild-type (wt) HeLa cells. Whole proteome mass spectrometric (MS) analysis of NCA-treated wt and KO cancer cells revealed regulated pathways and showed reduced levels of BST-2 upon NCA treatment. In-depth analysis of BST-2 levels in response to proteasome and lysosome inhibitors unraveled a lysosomal degradation path upon NCA treatment. As BST-2 mediates the release of epidermal growth factor receptor (EGFR) from lipid rafts to turn on proliferation signaling pathways, reduced BST-2 levels led to attenuated phosphorylation of STAT3. Furthermore, fluorescence microscopy confirmed increased colocalization of EGFR and lipid rafts in the presence of NCA. Overall, NCA represents a versatile anticancer natural product with a unique dual mode of action and unconventional inhibition of proliferation via BST-2 degradation.

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.

Rapid Diagnostic Platform for Personalized Vitamin B6 Detection in Erythrocytes via PLP Cofactor Mimics.

Personalized assessment of vitamin levels in point-of-care (POC) devices is urgently needed to advance the recognition of diseases associated with malnutrition and unbalanced diets. We here introduce a diagnostic platform, which showcases an easy and rapid readout of vitamin B6 (pyridoxal phosphate, PLP) levels in erythrocytes as a first step toward a home-use POC. The technology is based on fluorescent probes, which bind to PLP-dependent enzymes (PLP-DEs) and thereby indirectly report their occupancy with endogenous B6. For example, low vitamin levels result in high probe binding, yielding a strong signal and vice versa. Antibodies against signature human PLP-DEs were immobilized on microarrays to capture probe labeled enzymes for fluorescent detection. Calibrating the system with defined B6 levels revealed a concentration-depended readout as well as sufficient sensitivity for its detection in erythrocytes. To account for individual differences in protein expression, a second antibody was used to normalize protein abundance. This sandwiched assay correctly reported relative B6 levels in human erythrocyte samples, as confirmed by classical laboratory diagnostics. In principle, the platform layout can be easily expanded to other crucial vitamins beyond B6 via an analogous probe strategy.

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines.

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and ß subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate ß subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:ß assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

Intramembrane client recognition potentiates the chaperone functions of calnexin.

One-third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar-binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA.

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.

A Broad-Spectrum Antiviral Peptide Blocks Infection of Viruses by Binding to Phosphatidylserine in the Viral Envelope.

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors.

FICD activity and AMPylation remodelling modulate human neurogenesis.

Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signalling, localisation or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodelling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.

The Heat Shock Response in Yeast Maintains Protein Homeostasis by Chaperoning and Replenishing Proteins.

Life is resilient because living systems are able to respond to elevated temperatures with an ancient gene expression program called the heat shock response (HSR). In yeast, the transcription of hundreds of genes is upregulated at stress temperatures. Besides stress protection conferred by chaperones, the function of the majority of the upregulated genes under stress has remained enigmatic. We show that those genes are required to directly counterbalance increased protein turnover at stress temperatures and to maintain the metabolism. This anaplerotic reaction together with molecular chaperones allows yeast to efficiently buffer proteotoxic stress. When the capacity of this system is exhausted at extreme temperatures, aggregation processes stop translation and growth pauses. The emerging concept is that the HSR is modular with distinct programs dependent on the severity of the stress.

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2'-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

Surface topology affects wetting behavior of Bacillus subtilis biofilms.

The colonization of surfaces by bacterial biofilms constitutes a huge problem in healthcare and industry. When attempting biofilm inactivation or removal, it is crucial to sufficiently wet the biofilm surface with antibacterial agents; however, certain biofilms efficiently resist wetting, and the origin of this behavior remains to date unclear. Here, we demonstrate that, depending on the growth medium used, the model bacterium Bacillus subtilis can form biofilm colonies with distinct surface properties: we find either hydrophilic or two variants of hydrophobic behavior. We show that those differences in biofilm wetting correlate with distinct surface topologies which, in turn, give rise to different physical wetting regimes known from lotus leaves or rose petals. Forming biofilms with different wetting properties may help bacteria to survive in both arid and humid conditions. Furthermore, converting the surface polarity of a biofilm could facilitate their removal from surfaces by increasing their wettability.

Comparison of enzymatic properties and small molecule inhibition of γ-glutamyltranspeptidases from pathogenic and commensal bacteria.

Helicobacter pylori infects the stomach of 50% of the population worldwide, thus causing chronic gastritis. Although this infection can be cured by antibiotic treatment, therapeutic options are increasingly limited due to the development of resistances. The γ-glutamyl-transpeptidase (gGT) of H. pylori (HpgGT) is a virulence factor important for colonization and contributes to bacterial immune evasion. Therefore, this enzyme is a potential target for developing new anti-infectives. As species specificity of such compounds is required in order to avoid off-target or adverse effects, comparative analysis of the gGTs from different organisms is a prerequisite for drug development. To allow detailed biochemical and enzymatic characterization, recombinant gGTs from five different bacteria as well as Homo sapiens were characterized and compared. Investigation of the enzymatic activity, the binding modes of known inhibitors to the catalytic center, and a high resolution X-ray structure of the HpgGT provided a starting point for the identification of new inhibitory substances targeting HpgGT. Inhibitors with Ki values in the nm to mm range were identified and their binding modes were analyzed by mass spectrometry. The results of this study provide a basis for the development of species-specific lead compounds with anti-infective potential by effectively inhibiting HpgGT.

Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs.

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a ß-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.

ß-Sultams exhibit discrete binding preferences for diverse bacterial enzymes with nucleophilic residues.

ß-Sultams are potent electrophiles that modify nucleophilic residues in selected enzyme active sites. We here identify and characterize some of the specific bacterial targets and show a unique inhibition of the azoreductase family.

Target discovery of acivicin in cancer cells elucidates its mechanism of growth inhibition†Electronic supplementary information (ESI) available: Synthesis, cloning, protein expression, purification and biochemical assays. See DOI: 10.1039/c4sc02339k.

Acivicin is a natural product with diverse biological activities. Several decades ago its clinical application in cancer treatment was explored but failed due to unacceptable toxicity. The causes behind the desired and undesired biological effects have never been elucidated and only limited information about acivicin-specific targets is available. In order to elucidate the target spectrum of acivicin in more detail we prepared functionalized derivatives and applied them for activity based proteomic profiling (ABPP) in intact cancer cells. Target deconvolution by quantitative mass spectrometry (MS) revealed a preference for specific aldehyde dehydrogenases. Further in depth target validation confirmed that acivicin inhibits ALDH4A1 activity by binding to the catalytic site. In accordance with this, downregulation of ALDH4A1 by siRNA resulted in a severe inhibition of cell growth and might thus provide an explanation for the cytotoxic effects of acivicin.

Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT.

Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl) is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE). Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.

Methionine oxidation activates a transcription factor in response to oxidative stress.

Oxidant-mediated antibacterial response systems are broadly used to control bacterial proliferation. Hypochlorite (HOCl) is an important component of the innate immune system produced in neutrophils and specific epithelia. Its antimicrobial activity is due to damaging cellular macromolecules. Little is known about how bacteria escape HOCl-inflicted damage. Recently, the transcription factor YjiE was identified that specifically protects Escherichia coli from HOCl killing. According to its function, YjiE is now renamed HypT (hypochlorite-responsive transcription factor). Here we unravel that HypT is activated by methionine oxidation to methionine sulfoxide. Interestingly, so far only inactivation of cellular proteins by methionine oxidation has been reported. Mutational analysis revealed three methionines that are essential to confer HOCl resistance. Their simultaneous substitution by glutamine, mimicking the methionine sulfoxide state, increased the viability of E. coli cells upon HOCl stress. Triple glutamine substitution generates a constitutively active HypT that regulates target genes independently of HOCl stress and permanently down-regulates intracellular iron levels. Inactivation of HypT depends on the methionine sulfoxide reductases A/B. Thus, microbial protection mechanisms have evolved along the evolution of antimicrobial control systems, allowing bacteria to survive within the host environment.