Towards sequence-based principles for protein phase separation predictions

The phenomenon of protein phase separation, which underlies the formation of biomolecular condensates, has been associated with numerous cellular functions. Recent studies indicate that the amino acid sequences of most proteins may harbour not only the code for folding into the native state but also for condensing into the liquid-like droplet state and the solid-like amyloid state. Here we review the current understanding of the principles for sequence-based methods for predicting the propensity of proteins for phase separation. A guiding concept is that entropic contributions are generally more important to stabilise the droplet state than they are for the native and amyloid states. Although estimating these entropic contributions has proven difficult, we describe some progress that has been recently made in this direction. To conclude, we discuss the challenges ahead to extend sequence-based prediction methods of protein phase separation to include quantitative in vivo characterisations of this process.     

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires’ disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle–dependent manner is crucial for survival and replication. Here, we report that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on the protein homeostasis regulated by caseinolytic protease P (ClpP)-dependent proteolysis. We characterized the ClpP-dependent dynamic profiles of the regulatory and substrate proteins during the biphasic life cycle of L. pneumophila using proteomic approaches and discovered that ClpP-dependent proteolysis specifically and conditionally degraded the substrate proteins, thereby directly playing a regulatory role or indirectly controlling cellular events via the regulatory proteins. We further observed that ClpP-dependent proteolysis is required to monitor the abundance of fatty acid biosynthesis–related protein Lpg0102/Lpg0361/Lpg0362 and SpoT for the normal regulation of L. pneumophila differentiation. We also found that the control of the biphasic life cycle and bacterial virulence is independent. Furthermore, the ClpP-dependent proteolysis of Dot/Icm (defect in organelle trafficking/intracellular multiplication) type IVB secretion system and effector proteins at a specific phase of the life cycle is essential for bacterial pathogenesis. Therefore, our findings provide novel insights on ClpP-dependent proteolysis, which spans a broad physiological spectrum involving key metabolic pathways that regulate the transition of the biphasic life cycle and bacterial virulence of L. pneumophila, facilitating adaptation to aquatic and intracellular niches.     

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1^+/+ and Pnpla1^-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1^-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.     

CTP-controlled liquid–liquid phase separation of ParB

The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid–liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models.     

In Saccharomyces cerevisiae,withdrawal of the carbon source results in detachment of glycolytic enzymes from the cytoskeleton and in actin reorganization

Metabolons are dynamic associations of enzymes catalyzing consecutive reactions within a given pathway. Association results in enzyme stabilization and increased metabolic efficiency. Metabolons may use cytoskeletal elements, membranes and membrane proteins as scaffolds. The effects of glucose withdrawal on a putative glycolytic metabolon/F-actin system were evaluated in three Saccharomyces cerevisiae strains: a WT and two different obligate fermentative (OxPhos-deficient) strains, which obtained most ATP from glycolysis. Carbon source withdrawal led to inhibition of fermentation, decrease in ATP concentration and dissociation of glycolytic enzymes from F-actin. Depending on the strain, inactivation/reactivation transitions of fermentation took place in seconds. In addition, when ATP was very low, green fluorescent protein-labeled F-actin reorganized from highly dynamic patches to large, non-motile actin bodies containing proteins and enzymes. Glucose addition restored fermentation and cytoskeleton dynamics, suggesting that in addition to ATP concentration, at least in one of the tested strains, metabolon assembly/disassembly is a factor in the control of the rate of fermentation.     

Probing the sORF-Encoded Peptides of Deinococcus radiodurans in Response to Extreme Stress

Organisms have developed different mechanisms to respond to stresses. However, the roles of small ORF–encoded peptides (SEPs) in these regulatory systems remain elusive, which is partially because of the lack of comprehensive knowledge regarding these biomolecules. We chose the extremophile Deinococcus radiodurans R1 as a model species and conducted large-scale profiling of the SEPs related to the stress response. The integrated workflow consisting of multiple omics approaches for SEP identification was streamlined, and an SEPome of D. radiodurans containing 109 novel and high-confidence SEPs was drafted. Forty-four percent of these SEPs were predicted to function as antimicrobial peptides. Quantitative peptidomics analysis indicated that the expression of SEP068184 was upregulated upon oxidative treatment and gamma irradiation of the bacteria. SEP068184 was conserved in Deinococcus and exhibited negative regulation of oxidative stress resistance in a comparative phenotypic assay of its mutants. Further quantitative and interactive proteomics analyses suggested that SEP068184 might function through metabolic pathways and interact with cytoplasmic proteins. Collectively, our findings demonstrate that SEPs are involved in the regulation of oxidative resistance, and the SEPome dataset provides a rich resource for research on the molecular mechanisms of the response to extreme stress in organisms.     

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer–induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10–15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.     

Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

Fusarium oxysporum is one of the most abundant and diverse fungal species found in soils and includes nonpathogenic, endophytic, and pathogenic strains affecting a broad range of plant and animal hosts. Conidiation is the major mode of reproduction in many filamentous fungi, but the regulation of this process is largely unknown. Lysine acetylation (Kac) is an evolutionarily conserved and widespread posttranslational modification implicated in regulation of multiple metabolic processes. A total of 62 upregulated and 49 downregulated Kac proteins were identified in sporulating mycelia versus nonsporulating mycelia of F. oxysporum. Diverse cellular proteins, including glycolytic enzymes, ribosomal proteins, and endoplasmic reticulum–resident molecular chaperones, were differentially acetylated in the sporulation process. Altered Kac levels of three endoplasmic reticulum–resident molecular chaperones, PDI^K70, HSP70^K604, and HSP40^K32 were identified that with important roles in F. oxysporum conidiation. Specifically, K70 acetylation (K70ac) was found to be crucial for maintaining stability and activity of protein disulphide isomerase and the K604ac of HSP70 and K32ac of HSP40 suppressed the detoxification ability of these heat shock proteins, resulting in higher levels of protein aggregation. During conidial formation, an increased level of PDI^K70ac and decreased levels of HSP70^K604ac and HSP40^K32ac contributed to the proper processing of unfolded proteins and eliminated protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation in F. oxysporum.     

Deconvoluting the biology and druggability of protein lipidation using chemical proteomics

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes.     

New strategies for fluorescently labeling proteins in the study of amyloids

Amyloid proteins are widely studied, both for their unusual biophysical properties and their association with disorders such as Alzheimer’s and Parkinson’s disease. Fluorescence-based methods using site-specifically labeled proteins can provide information on the details of their structural dynamics and their roles in specific biological processes. Here, we describe the application of different labeling methods and novel fluorescent probe strategies to the study of amyloid proteins, both for in vitro biophysical experiments and for in vivo imaging. These labeling tools can be elegantly used to answer important questions on the function and pathology of amyloid proteins.     

Dynamic Refolding of OxyS sRNA by the Hfq RNA Chaperone

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.     

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO₂, formate, and H₂ that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially N^ε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, N^ε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.     

The effects of phytase transgenic maize on the community components and diversity of arthropods

It is important to understand the effect of phytase transgenic (PT) maize on arthropod communities in natural ecosystems. In this study, a 2-year survey of arthropod community biodiversity in fields of PT maize (0 7 8) and non-genetically modified (RA119, non-GM) maize was performed using sweep-net sampling on the stems and leaves of the maize plants. The results showed that there was no significant difference in the individual number of herbivorous, predatory, neutral and parasitic groups in PT maize and non-GM maize. The species number of herbivorous group in PT maize was significantly lower than that in non-GM maize in 2013 (p < 0.05). The proportions of different arthropod groups were almost identical in the PT maize and non-GM maize in terms of both species and individual number. Moreover, there was no significant difference in the Shannon-Weiner diversity index (H'), evenness index (J), dominance index (D), richness (S), and species abundance (N) between the two types of maize. The similarity coefficient of the arthropod community suggested that the arthropod community composition of PT maize was similar to that of non-GM maize. Furthermore, PT maize had no significant effect on the relative stability of the arthropod community. These results indicated that despite the presence of a relatively minor difference in arthropod community between the PT maize and non-GM maize, the PT maize had little effect on arthropod community biodiversity.     

Gut microbiome-derived glycine lipids are diet-dependent modulators of hepatic injury and atherosclerosis

Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr^?/? model of atherosclerosis. Intraperitoneal administration of L654 over 7 weeks to HFD-fed Ldlr^?/? mice resulted in hypocholesterolemic effects and significantly attenuated the progression of atherosclerosis. We found that L654 also reduced liver inflammatory and extracellular matrix gene expression, which may be related to inhibition of macrophage activation as demonstrated in vivo by lower major histocompatibility complex class II gene expression and confirmed in cell experiments. In addition, L654 and other bacterial glycine lipids in feces, liver, and serum were markedly reduced alongside changes in Bacteroidetes relative abundance in HFD-fed mice. Finally, we tested the bioactivities of L654 and related lipid 567 in chow-fed Apoe^?/? mice, which displayed much higher fecal glycine lipids relative to HFD-fed Ldlr^?/? mice. Administration of L654 or lipid 567 for 7 weeks to these mice reduced the liver injury marker alanine aminotransferase, but other effects seen in Ldlr^?/? were not observed. Therefore, we conclude that conditions in which gut microbiome-derived glycine lipids are lost, such as HFD, may exacerbate the development of atherosclerosis and liver injury, whereas correction of such depletion may protect from these disorders.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

The extraradical proteins of Rhizophagus irregularis: A shotgun proteomics approach

Arbuscular Mycorrhizal fungi (AMF, Glomeromycota) form obligate symbiotic associations with the roots of most terrestrial plants. Our understanding of the molecular mechanisms enabling AMF propagation and AMF-host interaction is currently incomplete. Analysis of AMF proteomes could yield important insights and generate hypotheses on the nature and mechanism of AMF-plant symbiosis. Here, we examined the extraradical mycelium proteomic profile of the arbuscular mycorrhizal fungus Rhizophagus irregularis grown on Ri T-DNA transformed Chicory roots in a root organ culture setting. Our analysis detected 529 different peptides that mapped to 474 translated proteins in the R. irregularis genome. R. irregularis proteome was characterized by a high proportion of proteins (9.9 % of total, 21.4 % of proteins with functional prediction) mediating a wide range of signal transduction processes, e.g. Rho1 and Bmh2, Ca-signaling (calmodulin, and Ca channel protein), mTOR signaling (MAP3K7, and MAPKAP1), and phosphatidate signaling (phospholipase D1/2) proteins, as well as members of the Ras signaling pathway. In addition, the proteome contained an unusually large proportion (53.6 %) of hypothetical proteins, the majority of which (85.8 %) were Glomeromycota-specific. Forty-eight proteins were predicted to be surface/membrane associated, including multiple hypothetical proteins of yet-unrecognized functions. However, no evidence for the overproduction of specific proteins, previously implicated in promoting soil health and aggregation was obtained. Finally, the comparison of R. irregularis proteome to previously published AMF proteomes identified a core set of pathways and processes involved in AMF growth. We conclude that R. irregularis growth on chicory roots requires the activation of a wide range of signal transduction pathways, the secretion of multiple novel hitherto unrecognized Glomeromycota-specific proteins, and the expression of a wide array of surface-membrane associated proteins for cross kingdom cell-to-cell communications.     

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg²⁺, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg²⁺. Two sABs from these selections, C12 and C18, showed different degrees of Mg²⁺-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg²⁺-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg²⁺-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg²⁺] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.