Jia-Xuan Chen

Herpes simplex virus type‐1 (HSV‐1) affects over 60% of the human population and increasingly develops resistance to antiviral therapies. Efficient HSV‐1 replication requires host‐derived deoxynucleotide triphosphates and the manipulation of cellular DNA replication and repair. This work positions the protein phosphatase 2A (PP2A) regulatory subunit PR130 (PPP2R3A) as cellular factor that suppresses the replication of laboratory strains and clinical isolates of HSV‐1 in epithelial and neuronal cells. HSV‐1 infection in turn decreases PR130 levels. Global proteome and phosphoproteome profiling combined with functional assays demonstrate that PR130 modulates key regulators of the cell cycle and DNA repair. PR130 controls the expression and phosphorylation of the cyclin‐dependent kinase (CDK) inhibitor p21 (CDKN1A) at serine 130 (S130) which CDK2 catalyzes. The levels and activities of p21, which HSV‐1 infection attenuates, and CDK2 are decisive factors for HSV‐1 replication. Inhibition of the ubiquitin‐specific protease USP7 stabilizes the p53‐p21 axis and reduces HSV‐1 viral titers. Additionally, PR130 depletion enhances signaling of the DNA damage‐responsive checkpoint kinase ataxia‐telangiectasia mutated (ATM) upon HSV‐1 infection and creates a dependency of HSV‐1 replication on ATM activity. These findings uncover host‐intrinsic mechanisms regulating HSV‐1 replication and highlight PR130 as central hub regulator of HSV‐1 infection.

SUMO, the small ubiquitin-like modifier, modulates interactions of folded proteins, but also affects the dynamics of biomolecular condensates. Moreover, SUMO can directly impinge on the biophysical properties of its targets, often increasing their solubility. TDP-43 is an RNA-binding protein containing an intrinsically disordered domain that participates in protein phase separation. Its aggregation, linked to neurodegenerative diseases, is counteracted by SUMOylation, but the underlying principles are poorly understood. We now reveal distinct mechanisms of how the two isoforms, SUMO1 and SUMO2, act on TDP-43. While SUMO1 inhibits phase separation by blocking TDP-43’s self-assembly site, SUMO2 enhances phase separation via proximity-induced self-interaction involving its N-terminus and promotes a liquid-like state that prevents aggregation. The latter represents a general principle by which SUMO solubilises other targets. Our findings provide insight into how SUMOylation regulates transitions between soluble, liquid-like and aggregated states.

Gene duplication underlies evolutionary innovation, yet many paralogues remain highly similar, raising questions about their functional divergence and physiological relevance. The spliceosomal Sm core protein SNRPB and its mammalian-specific paralogue SNRPN share over 90% sequence identity, but their distinct expression patterns - SNRPB being ubiquitous and SNRPN confined to the brain - suggest specialized functions. Why mammals have two different spliceosomes has remained obscure. Here, we generated isogenic human cell lines expressing ectopically either SNRPB or SNRPN exclusively and found that SNRPN stabilizes transcripts involved in energy metabolism and mitochondrial function, leading to increased mitochondrial abundance and oxygen consumption. Despite similar spliceosomal interactomes, SNRPN more strongly associates with the PRMT5 methylosome complex and exhibits dynamic arginine methylation in its C-terminal region that is sensitive to translation inhibition and amino acid availability. The SNRPN-dependent transcriptome responds to translation inhibition by stabilizing long, intron-rich genes involved in amino acid and energy metabolism. Our findings reveal a nutrient-sensitive, methylation-dependent mechanism that differentiates the two paralogues. This suggests that SNRPN functions as a metabolic-specialized spliceosomal subunit thereby providing tissue-specific adaptation of RNA processing in mammals.

Zusammenfassung: Das Protein Checkpoint‐Kinase‐1 (CHK1) steuert die Replikationsgeschwindigkeit und die Reparatur der DNA. Tumorzellen sind in ihrem Wachstum von CHK1 abhängig, dessen hohe Expression mit einer schlechteren Prognose von an Tumoren erkrankten Menschen assoziiert ist. Wir entdeckten ein Molekül, dass die Proteolyse von CHK1 auslösen kann (sogenanntes PROTAC für CHK1). Dieses PROTAC MA203 besteht aus dem Typ‐I‐Kinaseinhibitor Rabusertib, der bevorzugt aktiviertes CHK1 hemmt, sowie dem Cereblon‐Liganden Pomalidomid. MA203 beschleunigt den von dem Strukturprotein Cereblon (CRBN) abhängigen Abbau von CHK1 über den zellulären Proteasomen‐Komplex. Dies gilt für von soliden Tumoren isolierte Zellen und für akute Leukämiezellen. Durch Chemotherapie induzierter DNA‐Replikationsstress und die damit einhergehende Aktivierung von CHK1 fördern diesen Prozess, der DNA‐Schäden auslöst und Tumorzellen dem programmierten Zelltod (Apoptose) zuführt. Biochemische und zelluläre Bindungsstudien bestätigen die Potenz und Selektivität von MA203. Gesunde, reife und junge hämatopoetische Zellen, Stromazellen und Retina‐Epithelzellen werden jedoch nicht von MA203 geschädigt. MA203 ist seinem korrespondierenden Inhibitor hinsichtlich DNA‐Schädigung, Regulation von Apoptose‐regulierenden BCL2‐Proteinen und der Induktion von Apoptose in Tumorzellen überlegen. Diese Prozesse können unabhängig vom tumorsuppressiven Transkriptionsfaktor p53 erfolgen. Die Eliminierung des CHK1‐Proteins als Strukturelement ‐nicht jedoch seine Hemmung‐ löst eine proteasomale Degradation von DNA‐Replikationsproteinen und DNA‐Reparaturproteinen aus. Ein genetisch vermitteltes Ausschalten von CHK1 bestätigt, dass diese neu identifizierten Funktionen von CHK1 über seine bekannte katalytische Aktivität hinausgehen. Somit lassen sich neue Funktionen von CHK1 als Strukturelement der transformierten Zelle durch innovative pharmakologische Ansätze ausnutzen.

Checkpoint kinase‐1 (CHK1) controls DNA replication and repair. Tumor cells depend on CHK1, whose high levels are associated with worse patient prognosis. We define a bona fide proteolysis‐targeting‐chimera (PROTAC) for CHK1. PROTAC MA203 contains the type I kinase inhibitor rabusertib, which preferentially inhibits activated CHK1, and the cereblon (CRBN) ligand pomalidomide. MA203 accelerates CRBN‐dependent proteasomal degradation of CHK1 in solid tumor‐derived cells and acute leukemia cells. Chemotherapy‐induced DNA replication stress and a consequent activation of CHK1 accelerate this event‐driven process which promotes DNA damage and tumor cell apoptosis. Biochemical and cellular target engagement studies confirm the potency and selectivity of MA203. MA203 does not damage healthy differentiated and primitive hematopoietic cells, stromal cells, and retinal epithelial cells. MA203 is superior to its corresponding kinase inhibitor concerning DNA damage, dysregulation of BCL2 proteins, and apoptosis induction. These processes occur independently of the tumor‐suppressive transcription factor p53. Elimination of CHK1 protein as structural element, but not its inhibition per se, triggers a proteasomal degradation of key DNA replication and repair proteins. Genetic CHK1 elimination confirms that such newly recognized functions of CHK1 rely on functions beyond its well‐known catalytic activity. Thus, kinase‐independent functions of CHK1 can be exploited with innovative pharmacological agents.

Summary: The MRE11-RAD50-NBS1 (MRN) complex is a central, multifunctional factor in the detection, signaling and nucleolytic processing of DNA double-strand breaks (DSBs). To clarify how human MRN binds generic and telomeric DNA ends and can separate DNA end sensing from nuclease activities, we determined cryo-electron microscopy structures of human MRN bound to DNA and to DNA and the telomere protection factor TRF2. MRN senses DSBs through a tight clamp-like sensing state with closed coiled-coil domains, but auto-inhibited MRE11 nuclease. NBS1 wraps around the MRE11 dimer, with NBS1’s ATM recruitment motif sequestered by binding to the regulatory RAD50 S site, necessitating an allosteric switch for ATM activation. At telomeric DNA, TRF2 blocks the second S site via the iDDR motif to prevent nuclease and ATM activation. Our results provide a structural framework for topological DNA sensing and separation of sensing, signaling and processing activities of mammalian MRN. Highlights: - Human MRN senses DNA ends with an autoinhibited nuclease - NBS1’s C-terminus binds one RAD50 S site in the sensing state - TRF2 binds MRN’s second S site at telomeres - RAD50 and ATM compete for the NBS1 C-terminus

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system ofCaenorhabditis elegansto dissect the action of two virulence factors from its natural fungal pathogenDrechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.