Human shelterin protein POT1 prevents severe telomere instability induced by homology‐directed DNA repair

The evolutionarily conserved POT1 protein binds single‐stranded G‐rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R‐loops, and telomere fragility. We determine the telomeric proteome upon POT1‐loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1‐loss. Inactivation of the homology‐directed repair machinery suppresses POT1‐loss‐mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology‐directed repair.     

N‐glycosylation of PD‐1 promotes binding of camrelizumab

PD‐1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD‐1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD‐L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N‐glycosylation sites of PD‐1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N‐glycan composition in PD‐1, and show that the binding affinity of camrelizumab, a recently approved PD‐1‐specific MAb, to non‐glycosylated PD‐1 proteins from E. coli is substantially decreased compared with glycosylated PD‐1. The structure of the camrelizumab/PD‐1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD‐1, while the light chain sterically inhibits the binding of PD‐L1 to PD‐1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD‐L1 is substantially reduced for glycosylation‐deficient PD‐1. These results increase our understanding of how glycosylation affects the activity of PD‐1‐specific MAbs during immune checkpoint therapy.     

Long non‐coding RNA ELDR enhances oral cancer growth by promoting ILF3‐cyclin E1 signaling

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer with a 5‐year overall survival rate of 50%. Thus, there is a critical need to understand the disease process, and to identify improved therapeutic strategies. Previously, we found the long non‐coding RNA (lncRNA) EGFR long non‐coding downstream RNA (ELDR) induced in a mouse tongue cancer model; however, its functional role in human oral cancer remained unknown. Here, we show that ELDR is highly expressed in OSCC patient samples and in cell lines. Overexpression of ELDR in normal non‐tumorigenic oral keratinocytes induces cell proliferation, colony formation, and PCNA expression. We also show that ELDR depletion reduces OSCC cell proliferation and PCNA expression. Proteomics data identifies the RNA binding protein ILF3 as an interacting partner of ELDR. We further show that the ELDR‐ILF3 axis regulates Cyclin E1 expression and phosphorylation of the retinoblastoma (RB) protein. Intratumoral injection of ELDR‐specific siRNA reduces OSCC and PDX tumor growth in mice. These findings provide molecular insight into the role of ELDR in oral cancer and demonstrate that targeting ELDR has promising therapeutic potential.     

BAZ2A safeguards genome architecture of ground‐state pluripotent stem cells

Chromosomes have an intrinsic tendency to segregate into compartments, forming long‐distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground‐state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub‐domains within the active A compartment, which intersect through long‐range contacts. We found that ground‐state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A‐dependent manner. Finally, ground‐state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.     

The tRNA‐like small noncoding RNA mascRNA promotes global protein translation

mascRNA is a small cytoplasmic RNA derived from the lncRNA MALAT1. After being processed by the tRNA processing enzymes RNase P and RNase Z, mascRNA undergoes CCA addition like tRNAs and folds into a tRNA‐like cloverleaf structure. While MALAT1 functions in multiple cellular processes, the role of mascRNA was largely unknown. Here, we show that mascRNA binds directly to the multi‐tRNA synthetase complex (MSC) component glutaminyl‐tRNA synthetase (QARS). mascRNA promotes global protein translation and cell proliferation by positively regulating QARS protein levels. Our results uncover a role of mascRNA that is independent of MALAT1, but could be part of the molecular mechanism of MALAT1''s function in cancer, and provide a paradigm for understanding tRNA‐like structures in mammalian cells.     

Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2

Nucleosomes are dynamic entities with wide‐ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo‐EM structures of nucleosome core particles containing human H2A.B (H2A.B‐NCP) at atomic resolution, identifying large‐scale structural rearrangements in the histone octamer in H2A.B‐NCP. H2A.B‐NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left‐handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B‐specific residues, including a ROF (“regulating‐octamer‐folding”) sequence of six consecutive residues, are responsible for loosening of H2A.B‐NCPs. Unlike H2A.B‐NCP, the H2A.Z.2.2‐containing nucleosome (Z.2.2‐NCP) adopts a less‐extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2‐specific ROF in both H2A.Z.2.2‐NCP opening and SWR1‐dependent histone replacement. Taken together, these first high‐resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.     

Pseudo‐repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

Doublecortin (DCX) is a neuronal microtubule‐associated protein (MAP) indispensable for brain development. Its flexibly linked doublecortin (DC) domains—NDC and CDC—mediate microtubule (MT) nucleation and stabilization, but it is unclear how. Using high‐resolution time‐resolved cryo‐EM, we mapped NDC and CDC interactions with tubulin at different MT polymerization stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat within DCX appears to have a distinct role in MT nucleation and stabilization: CDC is a conformationally plastic module that appears to facilitate MT nucleation and stabilize tubulin–tubulin contacts in the nascent MT lattice, while NDC appears to be favored along the mature lattice, providing MT stabilization. Our structures of MT‐bound DC domains also explain in unprecedented detail the DCX mutation‐related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo‐repeats of tubulin/MT binding elements chaperone or stabilize distinct conformational transitions to regulate distinct stages of MT dynamic instability.     

Protein structure,amino acid composition and sequence determine proteome vulnerability to oxidation‐induced damage

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation‐resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ‐irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein‐intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties.     

NEDD4L‐mediated Merlin ubiquitination facilitates Hippo pathway activation

The tumor suppressor Merlin/NF2, a key activator of the Hippo pathway in growth control, is regulated by phosphorylation. However, it is uncertain whether additional post‐translational modifications regulate Merlin. Here, we show that ubiquitination is required to activate Merlin in the Hippo pathway. Ubiquitinated Merlin is mostly conjugated by one or two ubiquitin molecules. Such modification is promoted by serine 518 dephosphorylation in response to Ca²⁺ signaling or cell detachment. Merlin ubiquitination is mediated by the E3 ubiquitin ligase, NEDD4L, which requires a scaffold protein, AMOTL1, to approach Merlin. Several NF2‐patient‐derived Merlin mutations disrupt its binding to AMOTL1 and its regulation by the AMOTL1‐NEDD4L apparatus. Lysine (K) 396 is the major ubiquitin conjugation residue. Disruption of Merlin ubiquitination by the K396R mutation or NEDD4L depletion diminishes its binding to Lats1 and inhibits Lats1 activation. These effects are also accompanied by loss of Merlin''s anti‐mitogenic and tumor suppressive properties. Thus, we propose that dephosphorylation and ubiquitination compose an intramolecular relay to activate Merlin functions in activating the Hippo pathway during growth control.     

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss‐of‐function screenings with analysis of MT‐dynamics in human cells to investigate the molecular mechanisms underlying MT‐flux. We report that kinesin‐7/CENP‐E at kinetochores (KTs) is the predominant driver of MT‐flux in early prometaphase, while kinesin‐4/KIF4A on chromosome arms facilitates MT‐flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin‐5/EG5 and kinesin‐12/KIF15, and our data suggest that the MT‐flux driving force is transmitted from non‐KT‐MTs to KT‐MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT‐flux rate correlates with spindle length, and this correlation depends on the establishment of stable end‐on KT‐MT attachments. Strikingly, we find that MT‐flux is required to regulate spindle length by counteracting kinesin 13/MCAK‐dependent MT‐depolymerization. Thus, our study unveils the long‐sought mechanism of MT‐flux in human cells as relying on the coordinated action of four kinesins to compensate for MT‐depolymerization and regulate spindle length.     

A new l‐arginine oxidase engineered from l‐glutamate oxidase

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐arginine. The oxidative deamination activity of LGOX to l‐arginine is higher than that of l‐arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐arginine by LGOX R305E, is proportional to the concentration of l‐arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐arginine. Thus, LGOX R305E is suitable for the determination of l‐arginine.     

Kinetic characteristics of long‐term repeated fed‐batch (LtRFb) l‐lactic acid fermentation by a Bacillus coagulans strain

Application of degradable plastics is the most critical solution to plastic pollution. As the precursor of biodegradable plastic PLA (polylactic acid), efficient production of l‐lactic acid is vital for the commercial replacement of traditional plastics. Bacillus coagulans H‐2, a robust strain, was investigated for effective production of l‐lactic acid using long‐term repeated fed‐batch (LtRFb) fermentation. Kinetic characteristics of l‐lactic acid fermentation were analyzed by two models, showing that cell‐growth coupled production gradually replaces cell‐maintenance coupled production during fermentation. With the LtRFb strategy, l‐lactic acid was produced at a high final concentration of 192.7 g/L, on average, and a yield of up to 93.0% during 20 batches of repeated fermentation within 487.5 h. Thus, strain H‐2 can be used in the industrial production of l‐lactic acid with optimization based on kinetic modeling.