Max Perutz Labs

White spot syndrome virus (WSSV) infects crustaceans, causing severe losses in the global shrimp industry. Several properties, including the DNA genome sequence and the virion morphology, place WSSV as the single member of the Whispovirus genus of the Nimaviridae family. The DNA polymerase is one of the few gene products of the predicted 184 open reading frames to have been examined. Conserved sequence motifs found in many viral DNA polymerases are found in the WSSV DNA polymerase; nevertheless, the WSSV enzyme remains enigmatic, possessing over 1000 amino acids more than, for example, the DNA polymerase of herpes simplex virus 1 (HSV-1). To examine more closely the WSSV polymerase, we used AlphaFold to generate a structural model and compared it to the DNA polymerases of HSV-1, African swine fever virus and mpox virus. The exonuclease and polymerase domains of the WSSV enzyme were exactly defined based on the equivalence with the other viral enzymes; structurally, the WSSV enzyme appears most closely related to the HSV-1 enzyme. In contrast, the WSSV polymerase N-terminal domain showed an appreciably different architecture. However, the most unusual aspect of the WSSV polymerase is the C-terminal thumb domain which is modelled as two helical domains connected by a flexible acidic loop. This arrangement is quite unrelated to the thumb domains found in the other polymerases and is thus restricted to the WSSV enzyme. Given the uniqueness of such a vital cog of the replication machinery, it will be of interest to examine the structures of further WSSV proteins. (248 words).

Tetrahymena is a well-established ciliate model organism, known for its nuclear dualism and unique mechanism for transposon defense, wherein transposons are kept within the highly heterochromatinized micronucleus (MIC), which is transcriptionally inert except during meiosis. The discovery of preferential transcription in the meiotic MIC at regions enriched with transposons and the identification of MIC transcription regulatory proteins emphasize the necessity to elucidate the underlying mechanisms driving this process. In this study, we demonstrate that G/C-rich tracts are overrepresented in meiotic non-coding RNA (ncRNA) transcription regions and that RNA polymerase II (Pol II) is highly enriched at and around these tracts. Further analysis revealed that Pol II's association with G/C-rich tracts is not abolished in cells lacking Rib1, a Mediator complex-associated protein essential for MIC ncRNA biogenesis. Nevertheless, in the absence of Rib1, Pol II showed abnormal association with the meiotic MIC chromatin, suggesting that Rib1 is critical for maintaining Pol II's binding specificity. Through Rib1 truncation analysis, we found that the intrinsically disordered region, which contains putative phase-separating peptides, is crucial for its function. Disruption of phase-separation, either by deleting these peptides or by treating cells with a phase-separation disruption reagent, leads to aberrant localization of both Rib1 and Pol II on the MIC chromatin, implicating that Rib1 likely facilitates the MIC ncRNA transcription via phase-separation.

Somatic hypermutation (SHM) in variable regions of immunoglobulin genes by activation-induced deaminase (AID) is essential for the maturation of protective antibodies against pathogen and vaccine antigens. AID preferentially mutates cytosines within WRCH motifs (wherein W = A/T, R = A/G, and H = A/C/T) in single-stranded DNA, yet these motifs show large but reproducible variation in mutation frequency, suggesting a crucial role for sequences flanking the WRCH motifs (i.e., a sequence grammar) in determining mutational outcomes. However, the nature of this sequence grammar is poorly understood. Here, we demonstrate that identical sequence contexts can exert significantly varying effects on the mutagenesis of different WRCH motifs. Molecular dynamics simulations reveal that both the sequence context and the specific WRCH motif modulate AID activity by altering the mode and strength of AID’s interactions with single-stranded DNA. Repositioning a motif and its context within the variable region significantly alters its mutability. Therefore, the mutability of AID target cytosines is determined by a motif-specific sequence grammar that determines, in part, how activation-induced deaminase binds single-stranded DNA, as well as the motif position.

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN) is a C‐type lectin receptor expressed on antigen‐presenting cells, crucial for pathogen recognition and immune modulation. The shallow and polar carbohydrate binding site of DC‐SIGN presents challenges for ligand design. Here, we explored covalent modification targeting specific lysine residues as a novel strategy to modulate DC‐SIGN function. Screening a lysine‐targeted electrophilic fragment library using orthogonal functional assays identified two potent activators. Structural analyses via NMR spectroscopy, mass spectrometry and computational modeling confirmed structural perturbations of the carbohydrate recognition domain (CRD) and revealed distinct mechanisms of activation. While both activators significantly enhanced DC‐SIGN's affinity for monosaccharide ligands, one compound induced oligomerization via covalent coupling and non‐covalent secondary site interactions, whereas the other selectively modified lysine K373 directly within the primary carbohydrate binding site. These findings demonstrate the potential of lysine‐targeted covalent compounds as a novel therapeutic strategy for modulating DC‐SIGN function and potentially C‐type lectins in general.

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. By investigating chemical and genetic dependencies caused by mutations in methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 (MTHFD1), we discovered a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Genetic depletion and selective chemical degradation showed that a scaffolding role, rather than NUDT5 enzymatic activity, was causing this phenotype. NUDT5 interacted with phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme of purine de novo synthesis, to repress the pathway in response to increased purine abundance. Through this mechanism, loss of NUDT5 mediates resistance to purine analogs in cancer treatment and prevents adenosine toxicity in MTHFD1 deficiency.

The nuclear export of mRNA is an important step in eukaryotic gene expression1. Despite recent molecular insights into how newly transcribed mRNAs are packaged into ribonucleoprotein complexes (mRNPs)2,3, the subsequent events that govern mRNA export are poorly understood. Here we uncover the molecular basis underlying key events of human mRNA export, including the remodelling of mRNP-bound transcription-export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate their export. Our biochemical and structural data show that the ATPase UAP56 (also known as DDX39) acts as a central molecular switch that directs nucleoplasmic mRNPs from TREX to NPC-anchored TREX-2 complexes through its ATP-gated mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and evolutionarily conserved mRNAexport pathway.

Abstract Centrosomes are microtubule-organizing centers important for mitotic spindle assembly and chromosome segregation. During mitosis, centrosomes are exposed to mechanical forces via the microtubules they nucleate, yet the material properties underlying their response to these forces remain poorly understood. In this study, we systematically probed the mechanical behavior of C. elegans centrosomes, both in vitro and in vivo . Using microtubule perturbations and quantitative live cell imaging, we found that centrosomes become increasingly deformed during mitosis. Centrosome deformation is independent of cortical pulling forces but instead results from microtubule polymerization within the pericentriolar material. This deformation impacts centrosome size: as microtubule number decreases with cell volume in early cleavage divisions, centrosome size scales proportionately. To directly measure centrosome elasticity, we employed atomic force microscopy (AFM) on isolated centrosomes in vitro and Brillouin light scattering microscopy in developing embryos in vivo . Both approaches revealed that centrosomes progressively soften during mitosis. Theoretical modeling predicts that softening serves to dampen spindle force fluctuations, helping to protect kinetochore-microtubule interactions and safeguarding chromosome segregation. Further, softening may enhance centrosomal microtubule nucleation capacity, facilitating mitotic spindle assembly, particularly in large early embryonic cells. We propose that centrosome softening is a mechanical adaptation for mitosis that couples microtubule number to centrosome size through force-dependent deformation. This optimally balances two mitotic requirements: the need for robust microtubule nucleation and the ability to withstand spindle forces, thereby ensuring accurate cell division.

ABSTRACT Salinity, a major abiotic stressor, impacts protein trafficking and alters transport routes. The endosomal sorting complex required for transport (ESCRT)is a key player for membrane protein sorting and is increasingly recognized as pivotal element in abiotic stress responses. However, the functional role of ESCRT proteins in cereal crops under salt stress remains unexplored. Using the as near as possible to nature DRD-BIBLOX (Dark-Root device Brick Black Box), barley ( Hordeum vulgare , L.), although relatively salt-tolerant, exhibits impaired germination, seedling growth, and an altered root system architecture (RSA) under high salinity. Shotgun proteomic analysis revealed spatio-temporal regulation of ESCRT proteins in shoots and roots during seedling development. We identified HvSNF7.1 and HvVPS4 as critical, but differently acting modulators of salt stress responses in barley tissues. In this study we further reveal novel functions of ESCRT-0-like TOM-1-like proteins (TOLs): Previously described as gatekeepers of degradative protein sorting and as regulators of abscisic acid signaling, growth, and heat stress responses in Arabidopsis ( Arabidopsis thaliana ), HvTOLs had not been characterized for their role in salinity adaptation during plant development. Functional analyses demonstrated lethality in Hv toldouble and Hv tolquadruple mutants under standard growth conditions. In Arabidopsis, loss of At TOL3 alone impaired germination and root growth under salt stress, underscoring the essential role of individual TOL proteins in the spatio-temporal regulation of abiotic stress adaptation. These findings highlight ESCRT-mediated trafficking as a key determinant of cereal resilience under saline conditions and provide a molecular framework for improving crop stress tolerance.

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as an essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Cryo-electron microscopy analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3 MDa ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Our structure illustrates how UBR4 binds substrate and extends K48-specific ubiquitin chains. Efficient substrate targeting depends on both pre-ubiquitination and specific N-degrons, with KCMF1 acting as a key substrate filter. The architecture of the E4 megacomplex is conserved across eukaryotes, but species-specific adaptations allow UBR4 to perform its precisely tuned quality-control function in diverse cellular environments.