Biology subjects

Knowles, T. J.

Publications and source records attributed to Knowles, T. J..

6 recordsLinked to original sources

bioRxiv2022

The Role of UBE2-Conjugating Enzymes in the Mechanism of MuRF1 Ubiquitylation

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene)-type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with UBE2 (ubiquitin conjugating enzyme) to ubiquitylate its substrate protein. The understanding of MuRF1 function remains unclear as candidate UBE2 has not been elucidated and thus the mechanism of ubiquitylation is inconclusive. In the present study, we screened human ubiquitin dependent E2s using in-vitro ubiquitylation assays. We found that MuRF1 engages in ubiquitylation/auto-ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. Our result indicated that MuRF1 can cause mono-ubiquitylation, K48, and K63 specific poly-ubiquitin chains in a UBE2-dependent manner. Interestingly, we identified a two-step UBE2-dependent mechanism by which UBE2W allows MuRF1 to mono-ubiquitylate which then acts as an anchor for UBE2N/V and UBE2D generated poly-ubiquitin chain formation. Furthermore, MuRF1 was shown to cooperate with the identified interacting UBE2s to directly ubiquitylate substrates Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). Our work presents a novel insight into the mechanisms that underpin MuRF1 activity by highlighting the diversity of MuRF1 ubiquitylation enabled by different UBE2s.

molecular biology↗

bioRxiv2022

Peptidoglycan maturation controls spatiotemporal organisation of outer membrane proteins in Escherichia coli

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial to the maintenance of cellular integrity and enable survival in challenging environments1-5. The functionality of the outer membrane relies on outer membrane proteins (OMPs), which are inserted by the {beta}-barrel assembly machine, BAM6, 7. Previous work has shown that growing Escherichia coli cells segregate old OMPs towards the poles by an unknown mechanism8. Here, we demonstrate that peptidoglycan underpins the spatiotemporal organisation of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little impact on its activity, leading to preferential insertion of OMPs at division sites. Synchronising OMP biogenesis to cell wall growth enables bacteria to replenish their OMPs by binary partitioning. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation. This coordination offers new possibilities for the design of antibiotics that disrupt cell envelope integrity.

microbiology↗

bioRxiv2022

Modification of the SUMO activating enzyme subunit SAE2 directs SUMO isoform bias required for mitotic fidelity.

Mammalian cells possess three conjugatable SUMO variants: SUMO1 and the largely indistinguishable SUMO2 and SUMO3 (designated SUMO2/3). Some SUMOylated substrates are modified by both SUMO1 and SUMO2/3, while others show biased modifications towards SUMO1 or SUMO2/3. How preferential SUMO protein conjugation is coordinated is poorly understood. Here, we examine a modification of the catalytic component of the human SUMO Activation Enzyme, SAE2. We observe that lysine 164 of SAE2 is deacetylated during mitosis in an HDAC6-dependent manner. We find that an acetyl-analogue mutant, SAE2-K164Q, biases the activation and conjugation of SUMO2>SUMO1 and discriminates SUMO1 and SUMO2/3 through their C-terminal tails. Complementation of SAE2-depleted or inhibited cells with SAE2-K164Q restricts mitotic SUMO1-conjugates and increases multipolar spindle formation. We confirm the SUMO E1-dependent modification of the nuclear mitotic apparatus, NuMA, and find that the mitotic defects of both SAE2-K164Q complemented cells and HDAC6-inhibitor-treated cells are corrected by either over-expression of SUMO1 or by expression of a GFP-SUMO1-NuMA-K1766R fusion protein. Our observations suggest a model in which the SAE1:SAE2 enzyme is deacetylated on early mitosis to encourage the conjugation of SUMO1 to support mitotic fidelity. These surprising data reveal that the SUMO-activating enzyme can bias SUMO variant conjugation.

molecular biology↗

bioRxiv2022

The Vaccinia virus chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) - the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.

microbiology↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗

bioRxiv2020

MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria

MlaFEDB is a Gram-negative inner membrane protein complex involved in the inter membrane trafficking of phospholipids. Originally proposed to transport phospholipids in a retrograde direction, recent evidence suggests MlaFEDB may actually export phospholipids from the inner membrane to the periplasmic carrier protein, MlaC, potentially suggesting a role in either anterograde trafficking of phospholipids to the outer membrane or bidirectional phospholipid movement. MlaFEDB is part of the ABC transporter superfamily of proteins and has been shown to hydrolyse ATP through the cytoplasmic facing MlaF component. However, the movement of PLs from FEDB to MlaC has been shown to occur in an ATP independent fashion hence the role of ATP hydrolysis within this complex remains unclear. In this study we sought to elucidate the role of ATP and provide evidence to suggest MlaFEDB has flippase activity, utilising ATP hydrolysis to translocate phospholipids from the outer to the inner leaflet of the IM. We also show that in the absence of ATP MlaFEDB mediates the loading of MlaC with phospholipids directly from the inner leaflet only. Our data provides a novel role for MlaFEDB and presents a link between Mla driven phospholipid transport and ATP hydrolysis.

molecular biology↗