Rrm3 and Pif1 division of labor during replication through leading and lagging strand G-quadruplex.

Members of the conserved Pif1 family of 5'-3' DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve lagging strand G4s in vivo, little is known regarding Rrm3 function at G4s and its cooperation with Pif1 for G4 replication. Here, we monitored replication through G4 sequences in real time to show that Rrm3 is essential for efficient replisome progression through G4s located on the leading strand template, but not on the lagging strand. We found that Rrm3 importance for replication through G4s is dependent on its catalytic activity and its N-terminal unstructured region. Overall, we show that Rrm3 and Pif1 exhibit a division of labor that enables robust replication fork progression through leading and lagging strand G4s, respectively.

Developing a dual VEGF/PDL1 inhibitor based on high-affinity scFv heterodimers as an anti-cancer therapeutic strategy.

Cancer progression is enhanced by the interaction of programmed death-ligand 1 (PDL1), which is associated with inhibition of the immune response against tumors, and vascular endothelial growth factor (VEGF), which inhibits immune cell activity while inducing angiogenesis and proliferation of cancer cells. Dual inhibition of PDL1 and VEGF may therefore confer a synergistic anti-cancer therapeutic effect. We present a novel strategy for developing a therapeutic that simultaneously binds and inhibits both PDL1 and VEGF. We generated a bi-specific protein, designated DuRan-Bis, comprising a single chain variable fragment (scFv)-based inhibitor of PDL1 fused to an scFv-based inhibitor of VEGF, with the latter being attached to an Fc fragment. We found that DuRan-Bis binds to both PDL1 and VEGF with high affinity. Compared to treatments with mono-specific proteins, alone or in combination, the DuRan-Bis chimera showed superior inhibition of the proliferation of glioblastoma cells. In comparison to treatment with immune cells alone, a combination of immune cells with DuRan-Bis decreased the viability of head and neck cancer cells. To the best of our knowledge, this study is the first to use a single polypeptide chain scFv-scFv-Fc scaffold for engineering a high-affinity bi-specific inhibitor of PDL1 and VEGF.

Enhanced fluorescent imaging of proteins in live yeast cells using fluorescently labeled scFv.

Fluorescent labeling of proteins is a widespread approach for the microscopic examination of protein function, expression, and localization in the cell. Here, we present a protocol for the labeling of hemagglutinin (HA)-tagged protein of interest (POI) with the single-chain antibody (scFv) 2E2 fused to different fluorescent proteins (FPs) in Saccharomyces cerevisiae. We describe steps for expressing 2E2-FP, and HA tagging and labeling of POI. We detail in vivo fluorescent imaging of proteins at different cellular compartments and with diverse expression levels. For complete details on the use and execution of this protocol, please refer to Tsirkas et al. (2022).1.

Asgard ESCRT-III and VPS4 reveal conserved chromatin binding properties of the ESCRT machinery.

The archaeal Asgard superphylum currently stands as the most promising prokaryotic candidate, from which eukaryotic cells emerged. This unique superphylum encodes for eukaryotic signature proteins (ESP) that could shed light on the origin of eukaryotes, but the properties and function of these proteins is largely unresolved. Here, we set to understand the function of an Asgard archaeal protein family, namely the ESCRT machinery, that is conserved across all domains of life and executes basic cellular eukaryotic functions, including membrane constriction during cell division. We find that ESCRT proteins encoded in Loki archaea, express in mammalian and yeast cells, and that the Loki ESCRT-III protein, CHMP4-7, resides in the eukaryotic nucleus in both organisms. Moreover, Loki ESCRT-III proteins associated with chromatin, recruited their AAA-ATPase VPS4 counterpart to organize in discrete foci in the mammalian nucleus, and directly bind DNA. The human ESCRT-III protein, CHMP1B, exhibited similar nuclear properties and recruited both human and Asgard VPS4s to nuclear foci, indicating interspecies interactions. Mutation analysis revealed a role for the N terminal region of ESCRT-III in mediating these phenotypes in both human and Asgard ESCRTs. These findings suggest that ESCRT proteins hold chromatin binding properties that were highly preserved through the billion years of evolution separating Asgard archaea and humans. The conserved chromatin binding properties of the ESCRT membrane remodeling machinery, reported here, may have important implications for the origin of eukaryogenesis.

Transcription-replication coordination revealed in single live cells.

The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.

Protein fluorescent labeling in live yeast cells using scFv-based probes.

The fusion of fluorescent proteins (FPs) to endogenous proteins is a widespread approach for microscopic examination of protein function, expression, and localization in the cell. However, proteins that are sensitive to FP fusion or expressed at low levels are difficult to monitor using this approach. Here, we develop a single-chain fragment variable (scFv)-FP approach to efficiently label Saccharomyces cerevisiae proteins that are tagged with repeats of hemagglutinin (HA)-tag sequences. We demonstrate the successful labeling of DNA-binding proteins and proteins localized to different cellular organelles including the nuclear membrane, peroxisome, Golgi apparatus, and mitochondria. This approach can lead to a significant increase in fluorescence intensity of the labeled protein, allows C'-terminal labeling of difficult-to-tag proteins and increased detection sensitivity of DNA-damage foci. Overall, the development of a scFv-FP labeling approach in yeast provides a general and simple tool for the function and localization analysis of the yeast proteome.

A Bacterial Quorum Sensing Molecule Elicits a General Stress Response in Saccharomyces cerevisiae.

Bacteria assess their population density through a chemical communication mechanism termed quorum sensing, in order to coordinate group behavior. Most research on quorum sensing has focused primarily on its role as an intraspecies chemical signaling mechanism that enables the regulation of certain phenotypes through targeted gene expression. However, in recent years several seminal studies have revealed important phenomena in which quorum sensing molecules appear to serve additional roles as interspecies signals that may regulate microbial ecology. In this study, we asked whether the budding yeast Saccharomyces cerevisiae can sense chemical signals from prokaryotes. When exposed to a variety of quorum sensing molecules from different bacterial species and from Candida albicans we found that N-(3-oxododecanoyl)-L-homoserine lactone (C12) from the opportunistic human pathogen Pseudomonas aeruginosa induces a remarkable stress response in yeast. Microarray experiments confirmed and aided in interpreting these findings, showing a unique and specific expression pattern that differed significantly from the response to previously described stress factors. We further characterized this response and report preliminary findings on the molecular basis for the recognition of C12 by the yeast.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.

Cac1 WHD and PIP domains have distinct roles in replisome progression and genomic stability.

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells to maintain chromatin structure during DNA replication. The deposition of newly-synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. CAF-1 is an important histone chaperone complex and its main subunit, Cac1p, contains a PIP and WHD domain for interaction with PCNA and the DNA, respectively. While Cac1p subunit was extensively studied in different systems much less is known regarding the importance of the PIP and WHD domains in replication fork progression and genome stability. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in these Cac1p domains affect replication fork progression and post-replication characteristics. Our experiments revealed that mutations in the Cac1p WHD domain, which abolished the CAF-1-DNA interaction, slows down replication fork progression. In contrast, mutations in Cac1p PIP domain, abolishing Cac1p-PCNA interaction, lead to extended late-S/Anaphase duration, elevated number of RPA foci and increased spontaneous mutation rate. Our research shows that Cac1p WHD and PIP domains have distinct roles in high replisome progression and maintaining genome stability during cell cycle progression.

Human SIRT1 Multispecificity Is Modulated by Active-Site Vicinity Substitutions during Natural Evolution.

Many enzymes that catalyze protein post-translational modifications can specifically modify multiple target proteins. However, little is known regarding the molecular basis and evolution of multispecificity in these enzymes. Here, we used a combined bioinformatics and experimental approaches to investigate the evolution of multispecificity in the sirtuin-1 (SIRT1) deacetylase. Guided by bioinformatics analysis of SIRT1 orthologs and substrates, we identified and examined important amino acid substitutions that have occurred during the evolution of sirtuins in Metazoa and Fungi. We found that mutation of human SIRT1 at these positions, based on sirtuin orthologs from Fungi, could alter its substrate specificity. These substitutions lead to reduced activity toward K382 acetylated p53 protein, which is only present in Metazoa, without affecting the high activity toward the conserved histone substrates. Results from ancestral sequence reconstruction are consistent with a model in which ancestral sirtuin proteins exhibited multispecificity, suggesting that the multispecificity of some metazoan sirtuins, such as hSIRT1, could be a relatively ancient trait.

Application of directed evolution and back-to-consensus algorithms to human alpha1-antitrypsin leads to diminished anti-protease activity and augmented anti-inflammatory activities.

Primarily known as an elastase inhibitor, human alpha1-antitrypsin also exerts anti-inflammatory and immunomodulatory effects, both in vitro and in vivo. While the anti-protease mechanism of alpha1-antitrypsin is attributed to a particular protein domain coined the reactive center loop, anti-inflammatory and immunomodulatory loci within the molecule remain to be identified. In the present study, directed evolution and back-to-consensus algorithms were applied to human alpha1-antitrypsin. Six unique functional candidate sites were identified on the surface of the molecule; in manipulating these sites by point mutations, a recombinant mutant form of alpha1-antitrypsin was produced, depicting a requirement for sites outside the reactive center loop as essential for protease inhibition, and displaying enhanced anti-inflammatory activities. Taken together, outcomes of the present study establish a potential use for directed evolution in advancing our understanding of site-specific protein functions, offering a platform for development of context- and disease-specific alpha1-antitrypsin-based therapeutics.

Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures.

Pif1 DNA helicase is a potent unwinder of G-quadruplex (G4) structures in vitro and functions to maintain genome stability at G4 sequences in Saccharomyces cerevisiae. Here, we developed and utilized a live-cell imaging approach to quantitatively measure the progression rates of single replication forks through different G4 containing sequences in individual yeast cells. We show that in the absence of Pif1, replication rates through specific lagging strand G4 sequences in vivo is significantly decreased. In contrast, we found that in the absence of Pif1, replication rates through the same G4s on the leading strand are not decreased relative to the respective WT strains, showing that Pif1 is essential only for efficient replication through lagging strand G4s. Additionally, we show that a canonical PIP sequence in Pif1 interacts with PCNA and that replication through G4 structures is significantly slower in the absence of this interaction in vitro and in vivo. Thus, Pif1-PCNA interaction is essential for optimal replisome progression through G4 sequences, highlighting the importance of coupling between Pif1 activity and replisome progression during yeast genome replication.

A bi-specific inhibitor targeting IL-17A and MMP-9 reduces invasion and motility in MDA-MB-231 cells.

The cytokine IL-17A is associated with the progression of various cancers, but little is known about the molecular cross-talk between IL-17A and other tumor-promoting factors. Previous studies have shown that the IL-17A-mediated invasion of breast cancer cells can be inhibited by selective antagonists of the matrix metalloproteinase 9 (MMP-9), suggesting that the cross-talk between IL-17A and MMP-9 may promote cancer invasiveness and metastasis. Here, we present a novel strategy for developing cancer therapeutics, based on the simultaneous binding and inhibition of both IL-17A and MMP-9. To this end, we use a bi-specific heterodimeric fusion protein, comprising a natural inhibitor of MMPs (N-TIMP2) fused with an engineered extracellular domain (V3) of the IL-17A receptor. We show that, as compared with the mono-specific inhibitors of IL-17A (V3) and MMP-9 (N-TIMP2), the engineered bi-specific fusion protein inhibits both MMP-9 activation and IL-17A-induced cytokine secretion from fibroblasts and exhibits a synergistic inhibition of both the migration and invasion of breast cancer cells. Our findings demonstrate, for the first time, that dual targeting of inflammatory (IL-17A) and extracellular matrix remodeling (MMP) pathways can potentially be used as a novel therapeutic approach against cancer. Moreover, the platform developed here for generating the bi-specific IL-17A/MMP-9 inhibitor can be utilized for generating bi-specific inhibitors for other cytokines and MMPs.

A Live-Cell Imaging Approach for Measuring DNA Replication Rates.

We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level.

Point Mutation of a Non-Elastase-Binding Site in Human α1-Antitrypsin Alters Its Anti-Inflammatory Properties.

Introduction: Human α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357-366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo. Methods: His-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo. Results: Compared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding. Conclusion: Our data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes.

Enhanced PKMT-substrate recognition through non active-site interactions.

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in KM of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment.

Marker-free genetic manipulations in yeast using CRISPR/CAS9 system.

The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.

Directed evolution of SIRT6 for improved deacylation and glucose homeostasis maintenance.

Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.