Trifunctional fatty acid derivatives: the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

Trifunctional fatty acid derivatives demonstrate the impact of diazirine placement.

Functionalized lipid probes are a critical new tool to interrogate the function of individual lipid species, but the structural parameters that constrain their utility have not been thoroughly described. Here, we synthesize three palmitic acid derivatives with a diazirine at different positions on the acyl chain and examine their metabolism, subcellular localization, and protein interactions. We demonstrate that while they produce very similar metabolites and subcellular distributions, probes with the diazirine at either end pulldown distinct subsets of proteins after photo-crosslinking. This highlights the importance of thoughtful diazirine placement when developing probes based on biological molecules.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Trifunctional Sphinganine: A New Tool to Dissect Sphingolipid Function.

Functions and cell biology of the sphingolipids sphingosine and sphinganine in cells are not well understood. While some signaling roles for sphingosine have been elucidated, the closely related sphinganine has been described only insofar as it does not elicit many of the same signaling responses. Here, we prepared multifunctionalized derivatives of the two lipid species that differ only in a single double bond of the carbon backbone. Using these novel probes, we were able to define their spatiotemporal distributions within cells. Furthermore, we used these tools to systematically map the protein interactomes of both lipids. The lipid-protein conjugates, prepared through photo-crosslinking in live cells and extraction via click chemistry to azide beads, revealed significant differences in the captured proteins, highlighting their distinct roles in various cellular processes. This work elucidates mechanistic differences between these critical lipids and sets the foundation for further studies of the cellular functions of sphingosine and sphinganine.

Global Hfq-mediated RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery.

The RNA binding protein Hfq has a central role in the post-transcription control of gene expression in many bacteria. Numerous studies have mapped the transcriptome-wide Hfq-mediated RNA-RNA interactions in growing bacteria or bacteria that have entered short-term growth-arrest. To what extent post-transcriptional regulation underpins gene expression in growth-arrested bacteria remains unknown. Here, we used nitrogen (N) starvation as a model to study the Hfq-mediated RNA interactome as Escherichia coli enter, experience, and exit long-term growth arrest. We observe that the Hfq-mediated RNA interactome undergoes extensive changes during N starvation, with the conserved SdsR sRNA making the most interactions with different mRNA targets exclusively in long-term N-starved E. coli. Taking a proteomics approach, we reveal that in growth-arrested cells SdsR influences gene expression far beyond its direct mRNA targets. We demonstrate that the absence of SdsR significantly compromises the ability of the mutant bacteria to recover growth competitively from the long-term N-starved state and uncover a conserved post-transcriptional regulatory axis which underpins this process.

Flow in fetoplacental-like microvessels in vitro enhances perfusion, barrier function, and matrix stability.

Proper placental vascularization is vital for pregnancy outcomes, but assessing it with animal models and human explants has limitations. We introduce a 3D in vitro model of human placenta terminal villi including fetal mesenchyme and vascular endothelium. By coculturing HUVEC, placental fibroblasts, and pericytes in a macrofluidic chip with a flow reservoir, we generate fully perfusable fetal microvessels. Pressure-driven flow facilitates microvessel growth and remodeling, resulting in early formation of interconnected and lasting placental-like vascular networks. Computational fluid dynamics simulations predict shear forces, which increase microtissue stiffness, decrease diffusivity, and enhance barrier function as shear stress rises. Mass spectrometry analysis reveals enhanced protein expression with flow, including matrix stability regulators, proteins associated with actin dynamics, and cytoskeleton organization. Our model provides a powerful tool for deducing complex in vivo parameters, such as shear stress on developing vascularized placental tissue, and holds promise for unraveling gestational disorders related to the vasculature.

Dual truncation of tau by caspase-2 accelerates its CHIP-mediated degradation.

Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo.

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Lysosome-targeted multifunctional lipid probes reveal the sterol transporter NPC1 as a sphingosine interactor.

Lysosomes are catabolic organelles involved in macromolecular digestion, and their dysfunction is associated with pathologies ranging from lysosomal storage disorders to common neurodegenerative diseases, many of which have lipid accumulation phenotypes. The mechanism of lipid efflux from lysosomes is well understood for cholesterol, while the export of other lipids, particularly sphingosine, is less well studied. To overcome this knowledge gap, we have developed functionalized sphingosine and cholesterol probes that allow us to follow their metabolism, protein interactions, and their subcellular localization. These probes feature a modified cage group for lysosomal targeting and controlled release of the active lipids with high temporal precision. An additional photocrosslinkable group allowed for the discovery of lysosomal interactors for both sphingosine and cholesterol. In this way, we found that two lysosomal cholesterol transporters, NPC1 and to a lesser extent LIMP-2/SCARB2, bind to sphingosine and showed that their absence leads to lysosomal sphingosine accumulation which hints at a sphingosine transport role of both proteins. Furthermore, artificial elevation of lysosomal sphingosine levels impaired cholesterol efflux, consistent with sphingosine and cholesterol sharing a common export mechanism.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice.

The pruning of dendritic spines during development requires autophagy. This process is facilitated by long-term depression (LTD)-like mechanisms, which has led to speculation that LTD, a fundamental form of synaptic plasticity, also requires autophagy. Here, we show that the induction of LTD via activation of NMDA receptors or metabotropic glutamate receptors initiates autophagy in the postsynaptic dendrites in mice. Dendritic autophagic vesicles (AVs) act in parallel with the endocytic machinery to remove AMPA receptor subunits from the membrane for degradation. During NMDAR-LTD, key postsynaptic proteins are sequestered for autophagic degradation, as revealed by quantitative proteomic profiling of purified AVs. Pharmacological inhibition of AV biogenesis, or conditional ablation of atg5 in pyramidal neurons abolishes LTD and triggers sustained potentiation in the hippocampus. These deficits in synaptic plasticity are recapitulated by knockdown of atg5 specifically in postsynaptic pyramidal neurons in the CA1 area. Conducive to the role of synaptic plasticity in behavioral flexibility, mice with autophagy deficiency in excitatory neurons exhibit altered response in reversal learning. Therefore, local assembly of the autophagic machinery in dendrites ensures the degradation of postsynaptic components and facilitates LTD expression.

SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer's disease.

BACKGROUND: Human tauopathies including Alzheimer's disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. METHODS: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. RESULTS: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. CONCLUSIONS: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol.

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation-prone polyQ protein derived from human huntingtin. Expression of Q97-GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97-GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post-translational import of mitochondrial precursor proteins into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.

The Central Role of Salivary Metalloproteases in Host Acquired Resistance to Tick Feeding.

During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.

Stroma Transcriptomic and Proteomic Profile of Prostate Cancer Metastasis Xenograft Models Reveals Prognostic Value of Stroma Signatures.

Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential.

Pseudo-RNA-Binding Domains Mediate RNA Structure Specificity in Upstream of N-Ras.

RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Asparagine endopeptidase cleaves tau at N167 after uptake into microglia.

Tau cleavage by different proteolytic enzymes generates short, aggregation-prone fragments that have been implicated in the pathogenesis of Alzheimer's disease (AD). Asparagine endopeptidase (AEP) activity in particular has been associated with tau dysfunction and aggregation, and the activity of the protease is increased in both aging and AD. Using a mass spectrometry approach, we identified a novel tau cleavage site at N167 and confirmed its processing by AEP. In combination with the previously known site at N368, we show that AEP cleavage yields a tau fragment that is present in both control and AD brains at similar levels. AEP is a lysosomal enzyme, and our data suggest that it is expressed in microglia rather than in neurons. Accordingly, we observe tau cleavage at N167 and N368 after endocytotic uptake into microglia, but not neurons. However, tau168-368 does not accumulate in microglia and we thus conclude that the fragment is part of a proteolytic cascade leading to tau degradation. While we confirm previous studies showing increased overall AEP activity in AD brains, our data suggests that AEP-mediated cleavage of tau is a physiological event occurring during microglial degradation of the secreted neuronal protein. As a consequence, we caution against preventing AEP-mediated tau cleavage as a therapeutic approach in AD.