The role of p97 cofactors SPRTN and FAF1 in DNA replication

It is well known that cancer cells are loaded with excess proteins. To survive this proteotoxic crisis cancer cells rely heavily on the ubiquitin proteasome system (UPS). Valosin containing protein (VCP) or p97 has recently emerged as a central player of UPS. Moreover, p97 inhibitors were shown to possess potent anti-cancer properties. Owing to the essential function of p97, it can be assumed that general p97 inhibition will be equally toxic to surrounding normal tissues. Interestingly, p97 employs diverse sets of cofactors to govern distinct biological pathways. Thus understanding the p97-cofactor interaction in specific pathways may give us the ultimate edge by allowing us to abolish those cancer specific p97-cofactor interactions in our fight against cancer. This thesis explains novel function of two bonafide p97 cofactors SPRTN and FAF1 in DNA replication. Our discovery of a new human syndrome followed by its characterisation that monogenic and biallelic mutations in SPRTN leads to premature aging and juvenile hepatocellular carcinoma, have led us to identify the critical function of SPRTN in DNA replication. Our follow up work further revealed that SPRTN is a novel protease in humans and has the essential function in covalent DNA-protein crosslinks (DPCs) repair. To this end, we have found that SPRTN proteolytically removes CHK1 from chromatin, which is evolutionary conserved and vital for replication fork progression to preserve genomic integrity. Additionally, this thesis also explains a novel mechanism through which FAF1 downregulation in cancer cells might promote genomic instability. Our data supports a model where the p97-FAF1 complex binds to ubiquitinated CDT1 and degrades it via the proteasome after CDT1 mediated replication initiation, to prevent re-replication and genomic instability. The notion that high replication stress in some cancers can be utilised as an Achilles heel, led to the search for novel factors involved in DNA replication owing to its high therapeutic potential. Replication factors with enzymatic activities are especially appealing due to the relative ease of targeting them by small molecule inhibitors. This thesis thus highlights the great potential of the p97 systems in our battle against cancer.</p

The role of p97 cofactors SPRTN and FAF1 in DNA replication

It is well known that cancer cells are loaded with excess proteins. To survive this proteotoxic crisis cancer cells rely heavily on the ubiquitin proteasome system (UPS). Valosin containing protein (VCP) or p97 has recently emerged as a central player of UPS. Moreover, p97 inhibitors were shown to possess potent anti-cancer properties. Owing to the essential function of p97, it can be assumed that general p97 inhibition will be equally toxic to surrounding normal tissues. Interestingly, p97 employs diverse sets of cofactors to govern distinct biological pathways. Thus understanding the p97-cofactor interaction in specific pathways may give us the ultimate edge by allowing us to abolish those cancer specific p97-cofactor interactions in our fight against cancer. This thesis explains novel function of two bonafide p97 cofactors SPRTN and FAF1 in DNA replication. Our discovery of a new human syndrome followed by its characterisation that monogenic and biallelic mutations in SPRTN leads to premature aging and juvenile hepatocellular carcinoma, have led us to identify the critical function of SPRTN in DNA replication. Our follow up work further revealed that SPRTN is a novel protease in humans and has the essential function in covalent DNA-protein crosslinks (DPCs) repair. To this end, we have found that SPRTN proteolytically removes CHK1 from chromatin, which is evolutionary conserved and vital for replication fork progression to preserve genomic integrity. Additionally, this thesis also explains a novel mechanism through which FAF1 downregulation in cancer cells might promote genomic instability. Our data supports a model where the p97-FAF1 complex binds to ubiquitinated CDT1 and degrades it via the proteasome after CDT1 mediated replication initiation, to prevent re-replication and genomic instability. The notion that high replication stress in some cancers can be utilised as an Achilles heel, led to the search for novel factors involved in DNA replication owing to its high therapeutic potential. Replication factors with enzymatic activities are especially appealing due to the relative ease of targeting them by small molecule inhibitors. This thesis thus highlights the great potential of the p97 systems in our battle against cancer.</p

The role of p97 cofactors SPRTN and FAF1 in DNA replication

<p>It is well known that cancer cells are loaded with excess proteins. To survive this proteotoxic crisis cancer cells rely heavily on the ubiquitin proteasome system (UPS). Valosin containing protein (VCP) or p97 has recently emerged as a central player of UPS. Moreover, p97 inhibitors were shown to possess potent anti-cancer properties. Owing to the essential function of p97, it can be assumed that general p97 inhibition will be equally toxic to surrounding normal tissues. Interestingly, p97 employs diverse sets of cofactors to govern distinct biological pathways. Thus understanding the p97-cofactor interaction in specific pathways may give us the ultimate edge by allowing us to abolish those cancer specific p97-cofactor interactions in our fight against cancer. This thesis explains novel function of two bonafide p97 cofactors SPRTN and FAF1 in DNA replication.</p> <p>Our discovery of a new human syndrome followed by its characterisation that monogenic and biallelic mutations in SPRTN leads to premature aging and juvenile hepatocellular carcinoma, have led us to identify the critical function of SPRTN in DNA replication. Our follow up work further revealed that SPRTN is a novel protease in humans and has the essential function in covalent DNA-protein crosslinks (DPCs) repair. To this end, we have found that SPRTN proteolytically removes CHK1 from chromatin, which is evolutionary conserved and vital for replication fork progression to preserve genomic integrity.</p> <p>Additionally, this thesis also explains a novel mechanism through which FAF1 downregulation in cancer cells might promote genomic instability. Our data supports a model where the p97-^FAF1 complex binds to ubiquitinated CDT1 and degrades it via the proteasome after CDT1 mediated replication initiation, to prevent re-replication and genomic instability.</p> <p>The notion that high replication stress in some cancers can be utilised as an Achilles heel, led to the search for novel factors involved in DNA replication owing to its high therapeutic potential. Replication factors with enzymatic activities are especially appealing due to the relative ease of targeting them by small molecule inhibitors. This thesis thus highlights the great potential of the p97 systems in our battle against cancer.</p

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation

<p>Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.</p

Dynamics simulation of soybean agglutinin (SBA) dimer reveals the impact of glycosylation on its enhanced structural stability

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation. Recently, it has become clear that lectins exist as oligomers. Soybean agglutinin is a tetrameric legume lectin, each of whose sub-units are glycosylated. In the present study we explore the main origin for the stability of soybean agglutinin dimer. In order to understand the role of glycosylation on the dimeric interface, we have carried out normal (298K), high temperatures (380K, 500K) long explicit solvent molecular dynamics (MD) simulations and compared the structural and conformational changes between the glycosylated and non-glycosylated dimers. The study reveals that the high degree of stability at normal temperature is mostly contributed by interfacial ionic interactions (similar to 200 kcal/mol) between polar residues like Lys, Arg, Asp, Thr, Ser, Asn and Gln (62%). It maintains its overall folded conformation due to high subunit interactions at the non-canonical interface. Mainly five important hydrogen bonds between C=O of one beta sheet of one subunit with the N-H of other beta strand of the other subunit help to maintain the structural integrity. Ten inter subunit salt-bridge interactions between Arg 185-Asp192, Lys 163-Asp169, Asp 169-Lys 163 and Asp 192-Arg 185 at non-canonical interface appear to be important to maintain the three dimensional structure of SBA dimer. Moreover, our simulation results revealed that increase in vibrational entropy could decrease the free energy and contribute to the glycan-induced stabilization by similar to 45 kcal/mol at normal temperature. (C) 2016 Elsevier Ltd. All rights reserved

Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex

SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.ISSN:1362-4962ISSN:0301-561

Emerging structural details of transient amyloid-β oligomers suggest designs for effective small molecule modulators

Small oligomers are the major toxic species in many amyloid related diseases, but they are difficult to characterize and target. Here we construct tetra-peptides FXFX (X = F/K), designed to exploit cation-π, π-π and hydrophobic interactions to disrupt the critical F19-L34 contact recently found in Aβ₄₀ oligomers. FRFR accelerates Aβ₄₀ aggregation, and strongly inhibits its binding to lipid membranes, which is important in the context of toxicity. FKFK lacks both of these effects, which correlates with the weaker interaction of K with aromatic residues. Thus it appears possible to tune specific contacts in the oligomer and effectively change its properties

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatinassociated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging

Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.Peer reviewe