Nucleolar targeting in an early-branching eukaryote suggests a general physicochemical mechanism for ribosome protein sorting

The eukaryotic cell targets proteins to the organelles in which they function, both membrane-bound (like the nucleus) and non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals, and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in an early-branching eukaryote, Trypanosoma brucei, to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and protein physical properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins in comparison to mitochondrial ribosome proteins followed the same pattern - pointing to adaptation of physicochemical properties to assist segregation.

Identifying the Mechanism of Action of Bioactive 1,2-Cyclopropyl Carbohydrates

<p>Cyclopropanes and carbohydrates have long been used in the field of drug development. Previous work has shown that 1,2-cyclopropyl carbohydrates display bioactivity in both HeLa cancer cell lines¹ and in yeast² with a tentatively proposed mechanism of inhibition occurring through an enzymatic cyclopropane ring opening reaction and subsequent formation of a covalent bond with a target enzyme.² A small library of 1,2-cyclopropyl carbohydrate derivatives were synthesised based on known pharmacophores to examine further the potential mechanism of inhibition of such compounds and confirm the occurrence of enzyme-catalysed cyclopropane ring-opening reactions. Initial synthetic efforts were focused on the synthesis of the 1,2-dichlorocyclopropyl carbohydrate 23, which, through the optimisation of an essential C-6 detritylation reaction, was achieved in moderate yields of 32% over 7 steps. Following this, the ethoxycarbonyl substituted 1,2-cyclopropyl carbohydrate 54 was synthesised over 7 steps in a 22% yield through a rhodium acetate-catalysed addition of ethyl diazoacetate (49) to the glucal substrate 40. It was envisioned that if enzymatic cyclopropane ring-opening was occurring to form a C-7 carbanion, this would in turn be stabilised through the potential enolate formation of 54. Use of N,N-ditosylhydrazine in the synthesis of propargyl diazoacetate (58) followed by a rhodium acetate-catalysed cyclopropanation of 58 with substrate 40 resulted in the successful synthesis of 61 over 7 steps in a total yield of 9%. The incorporation of the propargyl substituent in 61 was introduced as a molecular probe in an attempt to isolate the target protein through an affinity purification procedure. The bioactivity of the propargyl derivative 61 was consistent with the synthesised compounds 23 and 54. It was proposed that these compounds undergo an enzymatic cyclopropane ring opening reaction accompanied with a clear diastereoselective preference for the α-stereoisomer of the cyclopropane ring, consistent with a target-based activation of the compounds. Chemical genetic analysis of the resulting bioactive compounds was undertaken using a deletion mutant array of Saccharomyces cerevisiae to elucidate a potential mechanism of action. Analysis of the results showed that, of the 4800 homozygous deletion strains tested in the high-throughput screens, a total of 122 strains were found following validation to sensitise and 68 to give resistance against 23 and 54. These sensitive and resistant mutants were subjected to a validation assay. Following validation, genes whose deletion led to sensitivity or resistance were then subjected to gene ontology term enrichment analysis which showed enrichment in the cytosolic ribosome, SNARE complex and SNAP receptor activity for resistant strains and enrichment in endoplasmic reticulum and endomembrane systems was found for the sensitive strain. Genes whose deletion sensitised to both compounds showed strong enrichment in cellular protein localisation, intra-golgi vesicale-mediated transport and the endomembrane system. Target identification and isolation were attempted through an affinity purification procedure using compound 61 and an azide-modified agarose resin. However, this was without success, either through inaccessibility of the alkyne of the target probe or because the target resides in the membrane-associated fraction which was discarded prior to treatment with the probe. This study suggests that the 1,2-cyclopropyl carbohydrates synthesised function through a cyclopropane ring-opening reaction, assisted by an enzymatic nucleophile. Chemical genetic analysis showed that the target of these compounds is involved in protein transport and protein localisation most likely relating to the vesicle tethering. Although many aspects of this work still need further investigation, either through the synthesis of new 1,2-cyclopropyl carbohydrates to increase bioactivity and better understand the enzymatic target, or through further biological procedures to better understand the mechanism of action, the use of 1,2-cyclopropyl carbohydrates as a potential pharmaceuticals or probes of protein trafficking shows much promise.</p>

The role of pro-domains in human growth factors and cytokines

Many growth factors and cytokines are produced as larger precursors, containing pro-domains, that require proteolytic processing to release the bioactive ligand. These pro-domains can be significantly larger than the mature domains and can play an active role in the regulation of the ligands. Mining the UniProt database, we identified almost one hundred human growth factors and cytokines with pro-domains. These are spread across several unrelated protein families and vary in both their size and composition. The precise role of each pro-domain varies significantly between the protein families. Typically they are critical for controlling bioactivity and protein localisation, and they facilitate diverse mechanisms of activation. Significant gaps in our understanding remain for pro-domain function — particularly their fate once the bioactive ligand has been released. Here we provide an overview of pro-domain roles in human growth factors and cytokines, their processing, regulation and activation, localisation as well as therapeutic potential.

In-Pero: Exploiting Deep Learning Embeddings of Protein Sequences to Predict the Localisation of Peroxisomal Proteins

Peroxisomes are ubiquitous membrane-bound organelles, and aberrant localisation of peroxisomal proteins contributes to the pathogenesis of several disorders. Many computational methods focus on assigning protein sequences to subcellular compartments, but there are no specific tools tailored for the sub-localisation (matrix vs. membrane) of peroxisome proteins. We present here In-Pero, a new method for predicting protein sub-peroxisomal cellular localisation. In-Pero combines standard machine learning approaches with recently proposed multi-dimensional deep-learning representations of the protein amino-acid sequence. It showed a classification accuracy above 0.9 in predicting peroxisomal matrix and membrane proteins. The method is trained and tested using a double cross-validation approach on a curated data set comprising 160 peroxisomal proteins with experimental evidence for sub-peroxisomal localisation. We further show that the proposed approach can be easily adapted (In-Mito) to the prediction of mitochondrial protein localisation obtaining performances for certain classes of proteins (matrix and inner-membrane) superior to existing tools.

The uniqueBrucellaeffectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. The mechanisms controlling nuclear protein localisation in the context of stress responses induced upon bacterial infection are still poorly understood. Here we show that theBrucella abortuseffectors NyxA and NyxB interfere with the host sentrin specific protease 3 (SENP3), which is essential for intracellular replication. Translocated Nyx effectors directly interact with SENP3viaa defined acidic patch identified from the crystal structure of NyxB, preventing its nucleolar localisation at the late stages of the infection. By sequestering SENP3, the Nyx effectors induce the cytoplasmic accumulation of the nucleolar AAA-ATPase NVL, the large subunit ribosomal protein L5 (RPL5) and the ribophagy receptor NUFIP1 in Nyx-enriched structures in the vicinity of replicating bacteria. This shuttling of ribosomal biogenesis-associated nucleolar proteins is negatively regulated by SENP3 and dependent on the autophagy-initiation protein Beclin1, indicative of a ribophagy-derived process induced duringBrucellainfection. Our results highlight a new nucleomodulatory function by two uniqueBrucellaeffectors, and reveal that SENP3 is a critical regulator of the subcellular localisation of multiple nucleolar proteins duringBrucellainfection, promoting intracellular replication.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells

Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised in the apical membrane of epithelial cells. Loss or mis-localisation of Crb is often associated with disruption of apico-basal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown here, accumulates in the oocyte of developing egg chambers. We narrowed down the Localization Element (LE) of crb mRNA to 47 nucleotides forming a putative stem-loop structure, suggesting to be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role of the Drosophila 3’-UTR in regulating translation in a tissue specific manner.

Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs

ABSTRACTProtein localisation in the cell is controlled through the function of trafficking receptors, which recognise specific signal sequences and direct cargo proteins to different locations. The KDEL receptor (KDELR) was one of the first intracellular trafficking receptors identified and plays an essential role in maintaining the integrity of the early secretory pathway. The receptor recognises variants of a canonical C-terminal Lys-Asp-Glu-Leu (KDEL) signal sequence on ER-resident proteins when these escape to the Golgi, and targets these proteins to COPI- coated vesicles for retrograde transport back to the ER. The empty receptor is then recycled from the ER back to the Golgi by COPII-coated vesicles. Crystal structures of the KDELR show that it is structurally related to the PQ-loop family of transporters that are found in both pro- and eukaryotes, and shuttle sugars, amino acids and vitamins across cellular membranes. Furthermore, analogous to PQ-loop transporters, the KDELR undergoes a pH-dependent and ligand-regulated conformational cycle. Here, we propose that the striking structural similarity between the KDELR and PQ-loop transporters reveals a connection between transport and trafficking in the cell, with important implications for understanding trafficking receptor evolution and function.