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The article examines studies on the mechanism of ribonucleic acid interference (RNAi)-dependent assembly of heterochromatin chromosome. It states that the chromosome is often linked with repetitive deoxyribonucleic acid (DNA) sequences. Proteins required for heterochromatic formation in the yeast, including Chp1, are cited. Also noted is that a biochemical analysis of the proteins was performed to define the complexes and molecular interactions that form and propagate such chromosomes.

We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Eukaryotic cells direct toxic misfolded proteins to various protein quality control pathways based on their chemical features and aggregation status. Aggregated proteins are targeted to selective autophagy or specifically sequestered into the "aggresome," a perinuclear inclusion at the microtubule-organizing center (MTOC). However, the mechanism for selectively sequestering protein aggregates into the aggresome remains unclear. To investigate aggresome formation, we reconstituted MTOC-directed aggregate transport in Xenopus laevis egg extract using AgDD, a chemically inducible aggregation system. High-resolution single-particle tracking revealed that dynein-mediated transport of aggregates was highly episodic, with average velocity positively correlated with aggregate size. Our mechanistic model suggests that the recurrent formation of the dynein transport complex biases larger aggregates towards the active transport state, compensating for the slowdown due to viscosity. Both episodic transport and positive size selectivity are specifically associated with aggresome-dynein adaptors. Coupling conventional dynein-activating adaptors to the aggregates perturbs aggresome formation and reverses size selectivity.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target., Competing Interests: A.E.P. is an employee of Ergon Pharmaceuticals Ltd. V.K.M. is a consultant to 5AM Ventures. A.M.M. has research funding from Janssen, Epizyme and Daiichi Sankyo. A.M.M. has consulted for Exo Therapeutics, Treeline Biosciences, Astra Zeneca, Epizyme. M.R.G reports research funding from Sanofi, Kite/Gilead, Abbvie and Allogene; consulting for Abbvie, Allogene and Bristol Myers Squibb; honoraria from BMS, Daiichi Sankyo and DAVA Oncology; and stock ownership of KDAc Therapeutics. All other authors declare no competing interests.

Activation of latent transforming growth factor (TGF)-β2 is incompletely understood. Unlike TGF-β1 and β3, the TGF-β2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-β2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVβ6 but not αVβ8. Both classes of motifs compete for the same binding site in αVβ6. Multiple changes in the longer motif underlie its specificity. ProTGF-β2 structures define interesting differences from proTGF-β1 and the structural context for activation by αVβ6. Some integrin-independent activation is also seen for proTGF-β2 and even more so for proTGF-β3. Our findings have important implications for therapeutics to αVβ6 in clinical trials for fibrosis, in which inhibition of TGF-β2 activation has not been anticipated.

The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair., (© 2022 by The American Society of Hematology.)

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis., (© 2021 Perez Verdaguer et al.)

Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

The testis transcriptome is exceptionally complex. Despite its complexity, previous testis transcriptome analyses relied on a reductive method for transcript identification, thus underestimating transcriptome complexity. We describe here a more complete testis transcriptome generated by combining Tuxedo, a reductive method, and spliced-RUM, a combinatorial transcript-building approach. Forty-two percent of the expanded testis transcriptome is composed of unannotated RNAs with novel isoforms of known genes and novel genes constituting 78 and 9.8% of the newly discovered transcripts, respectively. Across tissues, novel transcripts were predominantly expressed in the testis with the exception of novel isoforms which were also highly expressed in the adult ovary. Within the testis, novel isoform expression was distributed equally across all cell types while novel genes were predominantly expressed in meiotic and post-meiotic germ cells. The majority of novel isoforms retained their protein-coding potential while most novel genes had low protein-coding potential. However, a subset of novel genes had protein-coding potentials equivalent to known protein-coding genes. Shotgun mass spectrometry of round spermatid total protein identified unique peptides from four novel genes along with seven annotated non-coding RNAs. These analyses demonstrate the testis expresses a wide range of novel transcripts that give rise to novel proteins.

In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth--dependent signals control cell growth and the cell cycle., (© 2017 Clarke et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when coexpressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in coexpression pool varied from ∼1.5 to 5-fold compared to the batch mode, with up to ∼2.4 μM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ∼13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. This work also points to what is still needed to obtain self-replicating synthetic ribosomes in situ in the PURE system.