Proline-rich Domain Research Articles

Mxi1-0 Promotes Hypoxic Pulmonary Hypertension Via ERK/c-Myc-dependent Proliferation of Arterial Smooth Muscle Cells.

Background: Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality, and so far patients have failed to benefit from therapeutics clinically available. Max interacting protein 1–0 (Mxi1-0) is one of the functional isoforms of Mxi1. Although it also binds to Max, Mxi1-0, unlike other Mxi1 isoforms, cannot antagonize the oncoprotein c-Myc because of its unique proline rich domain (PRD). While Mxi1-0 was reported to promote cell proliferation via largely uncharacterized mechanisms, it is unknown whether and how it plays a role in the pathogenesis of HPH. Methods: GEO database was used to screen for genes involved in HPH development, and the candidate players were validated through examination of gene expression in clinical HPH specimens. The effect of candidate gene knockdown or overexpression on cultured pulmonary arterial cells, e.g., pulmonary arterial smooth muscle cells (PASMCs), was then investigated. The signal pathway(s) underlying the regulatory role of the candidate gene in HPH pathogenesis was probed, and the outcome of targeting the aforementioned signaling was evaluated using an HPH rat model. Results: Mxi1 was significantly upregulated in the PASMCs of HPH patients. As the main effector isoform responding to hypoxia, Mxi1-0 functions in HPH to promote PASMCs proliferation. Mechanistically, Mxi1-0 improved the expression of the proto-oncogene c-Myc via activation of the MEK/ERK pathway. Consistently, both a MEK inhibitor, PD98059, and a c-Myc inhibitor, 10058F4, could counteract Mxi1-0-induced PASMCs proliferation. In addition, targeting the MEK/ERK signaling significantly suppressed the development of HPH in rats. Conclusion: Mxi1-0 potentiates HPH pathogenesis through MEK/ERK/c-Myc-mediated proliferation of PASMCs, suggesting its applicability in targeted treatment and prognostic assessment of clinical HPH.

Dissecting the structural basis of Huntingtin pathogenesis: one molecule at the time

Huntington's disease (HD) arises from mutations in the Huntingtin protein (Htt) that expand its polyglutamine (polyQ) repeat beyond a threshold of 35 glutamines. This expansion which occurs in the exon 1 of Htt (Htt-Ex1) leads to aggregation, neuronal death, and HD. Htt-Ex1 consists of 3 parts: i) 17-amino acid N-terminal domain (N17), ii) polyQ tract, and iii) proline-rich domain (PRD). Evidence for HD and other neurodegenerative diseases suggest that aggregates may be inert/protective, while monomeric/oligomeric conformations are the toxic species.

The conformational dynamics of the flanking polyQ regions in the membrane-bound state of huntingtin exon 1

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin protein (httex1). The membrane interaction of httex1 is critical for its self-assembly and toxicity. While initial studies were essentially focused on polyQ peptide-lipid interaction, recent evidence supports that both flanking polyQ regions [the N17 segment and proline-rich domain (PRD)] strongly modulate the httex1 aggregation at membrane surfaces.

Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX

ALIX is involved in a myriad of essential cellular processes, most notably acting within the ESCRT pathway to mediate events such as cytokinesis and exosome biogenesis. Regulation of ALIX has been suggested to involve intramolecular, autoinhibitory interactions between individual ALIX domains. Interaction with the tyrosine kinase Src results in inactivation and redistribution of ALIX from late endosomal membranes to the cytosol, suggesting phosphorylation plays a role in ALIX autoinhibition. Yet, the molecular details of these interactions remain unclear owing to challenges relating to characterizing the large, disordered proline-rich domain (PRD) of ALIX.

RBM22, a Key Player of Pre-mRNA Splicing and Gene Expression Regulation, Is Altered in Cancer.

Simple SummaryRBM22 is a gene that encodes an essential RNA-Binding Protein involved in pre-mRNA splicing and transcription, with a DNA-binding function. This dual RNA/DNA-Binding activity provides a new insight of the regulation of gene expression by RBM22. Many studies have reported that RBM22 is essential for cell survival, mitosis, and differentiation processes. Consequently, RBM22 alterations are observed in several diseases and notably in cancer. It is suggested that RBM22 haploinsufficiency and dosage may be critical for its proper function. In this review, we aim at making a state-of-the-art review of this intriguing gene, encoding an RNA/DNA-binding protein that is understudied, and that could represent a potential therapeutic target in specific diseases and cancer.RNA-Binding Proteins (RBP) are very diverse and cover a large number of functions in the cells. This review focuses on RBM22, a gene encoding an RBP and belonging to the RNA-Binding Motif (RBM) family of genes. RBM22 presents a Zinc Finger like and a Zinc Finger domain, an RNA-Recognition Motif (RRM), and a Proline-Rich domain with a general structure suggesting a fusion of two yeast genes during evolution: Cwc2 and Ecm2. RBM22 is mainly involved in pre-mRNA splicing, playing the essential role of maintaining the conformation of the catalytic core of the spliceosome and acting as a bridge between the catalytic core and other essential protein components of the spliceosome. RBM22 is also involved in gene regulation, and is able to bind DNA, acting as a bona fide transcription factor on a large number of target genes. Undoubtedly due to its wide scope in the regulation of gene expression, RBM22 has been associated with several pathologies and, notably, with the aggressiveness of cancer cells and with the phenotype of a myelodysplastic syndrome. Mutations, enforced expression level, and haploinsufficiency of RBM22 gene are observed in those diseases. RBM22 could represent a potential therapeutic target in specific diseases, and, notably, in cancer.

Charge neutralization of lysine via carbamylation reveals hidden aggregation hot-spots in tau protein flanking regions.

Tau protein is found abundantly in neurofibrillary tangles in Alzheimer's disease (AD). The longest human tau isoform (2N4R) has 44 lysine residues. Several lysine-based post-translational modifications (PTMs) such as glycation, acetylation, ubiquitination, and sumoylation have been implicated not only in AD, but also in other tauopathies. Carbamylation is one such lysine neutralizing age-related nonenzymatic PTM which can modulate the aggregation propensity of tau. In this work, we have studied the aggregation potential of lysine-rich regions of tau upon carbamylation which do not aggregate in their native form. Using an array of biophysical and microscopic analyses, such as ThT kinetic assay, fluorescence microscopy, Congo red staining, and scanning electron microscopy, we demonstrate that peptides derived from four of five such regions exhibit robust fibrillar amyloid formation. These regions are found in the N-terminal projection domain that encompasses proline-rich domain (148-153 and 223-230), repeat domain R1 (253-260), as well as fibrillary core region (368-378), and can be described as hidden aggregation hot-spots which become activated upon carbamylation. We have further compared the impact of carbamylation with acetylation on the aggregation propensity of lysine-rich peptide (254 KKVAVV259 ) using biophysical experiments and molecular dynamics simulations and deduced that carbamylation is a much stronger driver of aggregation than acetylation. Our findings may offer more insight into amyloid fibrils' interaction with hidden aggregation-prone nucleating sequences that act as hot-spots for inducing tau fibrillation.

Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

Tumor suppressor p53, a critical regulator of cell fate, is frequently mutated in cancer. Mutation of p53 abolishes its tumor-suppressing functions or endows oncogenic functions. We recently found that p53 binds via its proline-rich domain to peptidase D (PEPD) and is activated when the binding is disrupted. The proline-rich domain in p53 is rarely mutated. Here, we show that oncogenic p53 mutants closely resemble p53 in PEPD binding but are transformed into tumor suppressors, rather than activated as oncoproteins, when their binding to PEPD is disrupted by PEPD knockdown. Once freed from PEPD, p53 mutants undergo multiple posttranslational modifications, especially lysine 373 acetylation, which cause them to refold and regain tumor suppressor activities that are typically displayed by p53. The reactivated p53 mutants strongly inhibit cancer cell growth in vitro and in vivo. Our study identifies a cellular mechanism for reactivation of the tumor suppressor functions of oncogenic p53 mutants.

Comprehensive Analysis of Hepatitis Delta Virus Assembly Determinants According to Genotypes: Lessons From a Study of 526 Hepatitis Delta Virus Clinical Strains.

Human hepatitis Delta virus (HDV) infection is associated to the most severe viral hepatic disease, including severe acute liver decompensation and progression to cirrhosis, and hepatocellular carcinoma. HDV is a satellite of hepatitis B virus (HBV) that requires the HBV envelope proteins for assembly of HDV virions. HDV and HBV exhibit a large genetic diversity that extends, respectively to eight (HDV-1 to -8) and to ten (HBV/A to/J) genotypes. Molecular determinants of HDV virion assembly consist of a C-terminal Proline-rich domain in the large Hepatitis Delta Antigen (HDAg) protein, also known as the Delta packaging domain (DPD) and of a Tryptophan-rich domain, the HDV matrix domain (HMD) in the C-terminal region of the HBV envelope proteins. In this study, we performed a systematic genotyping of HBV and HDV in a cohort 1,590 HDV-RNA-positive serum samples collected between 2001 to 2014, from patients originated from diverse parts of the world, thus reflecting a large genetic diversity. Among these samples, 526 HBV (HBV/A, B, C, D, E, and G) and HDV (HDV-1, 2, 3, and 5 to -8) genotype couples could be obtained. We provide results of a comprehensive analysis of the amino-acid sequence conservation within the HMD and structural and functional features of the DPD that may account for the yet optimal interactions between HDV and its helper HBV.

Wnt5a Induces ROR1 to Interact Grb2 to Enhance Ras Activation in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen found on chronic lymphocytic leukemia (CLL) cells, but not on healthy postpartum tissues. In prior studies we found that higher-level CLL-cell expression of ROR1 associated with accelerated disease progression and unfavorable clinical outcome (Cui B. et al., Blood, 128:2931, 2016). ROR1 is a receptor for Wnt5a, which is found at high levels in plasma of patients with CLL. We found Wnt5a could enhance ERK1/2 phosphorylation and leukemia-cell proliferation (Hasan MK. et al., Leukemia, 35:1621, 2021). Moreover, Wnt5a-enhanced ERK1/2 phosphorylation and leukemia-cell proliferation could be inhibited by UC-961, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain that is undergoing phase II clinical testing (Choi MY. et al., Cell Stem Cell, 22:951, 2018). However, it is not known how Wnt5a/ROR1-signaling induces ERK1/2 activation. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified Grb2 (Growth Factor Receptor Bound Protein 2), a cytoplasmic protein that may interact with SOS (son of sevenless) to induce activation of Ras, which can activate the mitogen-activated protein kinase (MAPK) pathway, leading to activation of ERK1/2. We found that treatment of serum-starved CLL cells with Wnt5a induced Grb2 to associate with ROR1 and activate Ras and ERK1/2; these effects could be blocked by siRNA silencing of Grb2 or by treatment with UC-961, indicating that Wnt5a induced activation of ERK1/2 is dependent upon expression of ROR1 and Grb2. To examine the structure-function relationship(s) in the interaction(s) between ROR1 and Grb2, we used the CLL-derived leukemia cell line MEC1, which we found expresses high-levels of Wnt5a, but not ROR1. Subclones of MEC1 cells transfected to express ROR1 (MEC1-ROR1) had significantly higher levels of ERK1/2 activation than MEC1 cells; immune-precipitation studies confirmed constitutive association of Grb2 and SOS1 with ROR1. This was not observed in MEC1-ROR1-Wnt5a -/- cells in which we disrupted both alleles of WNT5a via CRISPR-Cas9. Moreover, MEC1-ROR1 cells, or MEC1-ROR1-Wnt5a -/- cells treated with Wnt5a, had significantly higher levels of activated Ras and ERK1/2 than MEC1, MEC1-Wnt5a -/-, or MEC1-ROR1-Wnt5a -/-, demonstrating that activation of Ras, MAPK, and ERK1/2 in MEC1 was dependent upon on expression of ROR1 and Wnt5a. We next examined single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the proline-rich domain (PRD) of ROR1 at positions 784, 808, 826 or 841. Interestingly we found that P808A of ROR1 (MEC1-ROR1 P808A), a binding site of DOCK2 and critical for activation of ERK1/2, failed to complex with Grb2, SOS1, or enhance activation of Ras compared to MEC1-ROR1 cells. Overall, our results indicate that, upon Wnt5a stimulation, a novel complex of DOCK2/Grb2/SOS1 may form at position 808 of ROR1 that leads to activation of Ras and ERK1/2, which can promote proliferation of CLL cells and potentially contribute to the noted unfavorable prognostic implication of high-level expression of ROR1 in CLL. DisclosuresKipps: VelosBio, Inc.: Research Funding; Celgene: Honoraria, Research Funding; Genentech/Roche: Honoraria; Pharmacyclics/AbbiVie: Honoraria, Research Funding; Oncternal Therapeutics, Inc.: Current equity holder in publicly-traded company, Patents & Royalties: Cirmtuzumab, Research Funding; Janssen, Gilead, Dava Oncology,: Honoraria; Breast Cancer Research Foundation: Honoraria, Research Funding; Md Anderson Cancer Center: Research Funding; Gilead: Honoraria; National Cancer Institute/NIH: Honoraria, Research Funding; OncLive: Honoraria; Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS): Research Funding; California Institute for Regenerative Medicine (CIRM): Research Funding; European Research Initiative on CLL (ERIC): Honoraria; Dava Oncology: Honoraria; Patient Power, LLC: Honoraria; iwNHL: Honoraria; NCCN CLL/SLL Hairy Cell Leukemia Panel Meeting: Honoraria.

Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like β-sheet–rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B–mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.

An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics

PurposeSpatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear.MethodsThe clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells.ResultsWe found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion.ConclusionsOur data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Amplification of neurotoxic HTTex1 assemblies in human neurons

Huntington’s disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.

Distinct RNA polymerase transcripts direct the assembly of phase-separated DBC1 nuclear bodies in different cell lines.

The mammalian cell nucleus is a highly organized organelle that contains membrane-less structures referred to as nuclear bodies (NBs). Some NBs carry specific RNA types that play architectural roles in their formation. Here, we show two types of RNase-sensitive DBC1-containing NBs, DBC1 nuclear body (DNB) in HCT116 cells and Sam68 nuclear body (SNB) in HeLa cells, that exhibit phase-separated features and are constructed using RNA polymerase I or II transcripts in a cell type–specific manner. We identified additional protein components present in DNB by immunoprecipitation–mass spectrometry, some of which (DBC1 and heterogeneous nuclear ribonucleoprotein L [HNRNPL]) are required for DNB formation. The rescue experiment using the truncated HNRNPL mutants revealed that two RNA-binding domains and intrinsically disordered regions of HNRNPL play significant roles in DNB formation. All these domains of HNRNPL promote in vitro droplet formation, suggesting the need for multivalent interactions between HNRNPL and RNA as well as proteins in DNB formation.

Activity-dependent release of phosphorylated human tau from Drosophila neurons in primary culture

Neuronal activity can enhance tau release and thus accelerate tauopathies. This activity-dependent tau release can be used to study the progression of tau pathology in Alzheimer's disease (AD), as hyperphosphorylated tau is implicated in AD pathogenesis and related tauopathies. However, our understanding of the mechanisms that regulate activity-dependent tau release from neurons and the role that tau phosphorylation plays in modulating activity-dependent tau release is still rudimentary. In this study, Drosophila neurons in primary culture expressing human tau (hTau) were used to study activity-dependent tau release. We found that hTau release was markedly increased by 50 mM KCl treatment for 1 h. A similar level of release was observed using optogenetic techniques, where genetically targeted neurons were stimulated for 30 min using blue light (470 nm). Our results showed that activity-dependent release of phosphoresistant hTauS11A was reduced when compared with wildtype hTau. In contrast, release of phosphomimetic hTauE14 was increased upon activation. We found that released hTau was phosphorylated in its proline-rich and C-terminal domains using phosphorylation site-specific tau antibodies (e.g., AT8). Fold changes in detectable levels of total or phosphorylated hTau in cell lysates or following immunopurification from conditioned media were consistent with preferential release of phosphorylated hTau after light stimulation. This study establishes an excellent model to investigate the mechanism of activity-dependent hTau release and to better understand the role of phosphorylated tau release in the pathogenesis of AD since it relates to alterations in the early stage of neurodegeneration associated with increased neuronal activity.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella.

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of EkMasc and/or EkMascB in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Disease-associated mutations affect TIA1 phase separation and aggregation in a proline-dependent manner

T-cell restriction intracellular antigen 1 (TIA1) is an RNA-binding protein that is a major component of stress granules (SGs). The low complexity domain (LCD) of TIA1 plays a central role in facilitating SGs assembly through liquid–liquid phase separation (LLPS). Disruption of the LLPS process has been associated with several diseases. It has recently been shown that the proline-rich domain affects the LLPS process of some proteins (such as UBQLN2 and Tau). Thus, proline may regulate LLPS. The LCD of TIA1 contains 11 proline residues, and several proline-related mutations have been shown to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we demonstrated that TIA1 can undergo phase separation in cells. Additionally, disease-associated proline-to-leucine (P-L) mutations, which altered droplet morphology, facilitated the liquid-to-solid phase transition of TIA1 into solid-like amyloid fibrils. The changes in the physical properties of the P-L mutation altered the behavior of TIA1 in vivo and led to abnormal SGs kinetics, resulting in the formation of the pathological inclusions of ALS. Prolines are the key residues for regulating the LLPS of TIA1.