Transitional basal cells at the squamous-columnar junction generate Barrett's oesophagus.

In several organ systems, the transitional zone between different types of epithelium is a hotspot for pre-neoplastic metaplasia and malignancy, but the cells of origin for these metaplastic epithelia and subsequent malignancies remain unknown. In the case of Barrett's oesophagus, intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells. On the basis of a number of experimental models, several alternative cell types have been proposed as the source of this metaplasia but in all cases the evidence is inconclusive: no model completely mimics Barrett's oesophagus in terms of the presence of intestinal goblet cells. Here we describe a transitional columnar epithelium with distinct basal progenitor cells (p63+KRT5+KRT7+) at the squamous-columnar junction of the upper gastrointestinal tract in a mouse model. We use multiple models and lineage tracing strategies to show that this squamous-columnar junction basal cell population serves as a source of progenitors for the transitional epithelium. On ectopic expression of CDX2, these transitional basal progenitors differentiate into intestinal-like epithelium (including goblet cells) and thereby reproduce Barrett's metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues (including the anorectal junction) as well as in the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (believed to be a precursor of Barrett's oesophagus) are both characterized by the expansion of the transitional basal progenitor cells. Our findings reveal a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+KRT5+KRT7+ basal cells in this zone are the cells of origin for multi-layered epithelium and Barrett's oesophagus.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition.

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair.

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Mitosis-specific MRN complex promotes a mitotic signaling cascade to regulate spindle dynamics and chromosome segregation.

The MRE11-RAD50-NBS1 (MRN) complex is well known for participating in DNA damage response pathways in all phases of cell cycle. Here, we show that MRN constitutes a mitosis-specific complex, named mMRN, with a protein, MMAP. MMAP directly interacts with MRE11 and is required for optimal stability of the MRN complex during mitosis. MMAP colocalizes with MRN in mitotic spindles, and MMAP-deficient cells display abnormal spindle dynamics and chromosome segregation similar to MRN-deficient cells. Mechanistically, both MMAP and MRE11 are hyperphosphorylated by the mitotic kinase, PLK1; and the phosphorylation is required for assembly of the mMRN complex. The assembled mMRN complex enables PLK1 to interact with and activate the microtubule depolymerase, KIF2A, leading to spindle turnover and chromosome segregation. Our study identifies a mitosis-specific version of the MRN complex that acts in the PLK1-KIF2A signaling cascade to regulate spindle dynamics and chromosome distribution.

Metathesis Cascade-Triggered Depolymerization of Enyne Self-Immolative Polymers*.

A novel class of enyne self-immolative polymers (SIPs) capable of metathesis cascade-triggered depolymerization is reported. Studies on model compounds established 1,6-enyne structures for efficient metathesis cascade reactions. SIPs incorporating the optimized 1,6-enyne motif were prepared via both polycondensation and iterative exponential growth approaches. These SIPs demonstrated excellent stability in strong acid, base, nucleophiles, or at elevated temperatures, and can undergo efficient and complete depolymerization once triggered by a metathesis catalyst. Further studies revealed that introducing a terminal alkene to the chain end of the enyne SIPs improved the depolymerization efficiency, and established their potential as stimuli-responsive materials.

The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

PTPN14 is required for the density-dependent control of YAP1.

Through an shRNA-mediated loss-of-function screen, we identified PTPN14 as a potential tumor suppressor. PTPN14 interacts with yes-associated protein 1 (YAP1), a member of the hippo signaling pathway. We showed that PTPN14 promotes the nucleus-to-cytoplasm translocation of YAP1 during contact inhibition and thus inhibits YAP1 transactivation activity. Interestingly, PTPN14 protein stability was positively controlled by cell density. We identified the CRL2(LRR1) (cullin2 RING ubiquitin ligase complex/leucine-rich repeat protein 1) complex as the E3 ligase that targets PTPN14 for degradation at low cell density. Collectively, these data suggest that PTPN14 acts to suppress cell proliferation by promoting cell density-dependent cytoplasmic translocation of YAP1.

4-Hydroxyproline-Derived Sustainable Polythioesters: Controlled Ring-Opening Polymerization, Complete Recyclability, and Facile Functionalization.

The sustainable production of chemically recyclable polymers presents a significant opportunity to polymer scientists to tackle the growing environmental and energy problems of current petroleum-based plastics. Despite recent advances, however, there are still pressing needs for an expanded horizon of chemically recyclable polymers. Herein, we introduce a new paradigm of biosourced polythioesters (PTEs) with high polymerizability and complete recyclability under mild and economical conditions. The thiolactone monomers with a high ring strain can be easily prepared in a two-step process from 4-hydroxyproline. Controlled ring-opening polymerizations (ROP) using inexpensive and weak bases afford PTEs with high molar masses ( Mn) up to 259 kg mol-1 and narrow dispersities generally below 1.15. The properties of PTEs can be readily adjusted by copolymerization and/or pre/post-functionalization on the side chains. Selective and complete depolymerizations of the PTEs in dilute solution at ambient to modest temperatures recycle clean monomers. Density functional theory (DFT) calculation of model reactions provides mechanistic insights and highlights the importance of judicious molecular design. Taken together, the unique ROP/depolymerization chemistry of such PTEs may offer a sustainable solution for creating and manufacturing high-value materials such as optical/photochemical plastics, self-immolative polymers, and degradable biomaterials under situations where recycle and reuse are indispensable.

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.

Macrocyclization of Interferon-Poly(α-amino acid) Conjugates Significantly Improves the Tumor Retention, Penetration, and Antitumor Efficacy.

Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EG3Glu)20-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, N-P(EG3Glu)20-IFN, C-IFN-P(EG3Glu)20, and C-IFN-PEG. Our results found circ-P(EG3Glu)20-IFN to show considerably greater stability, binding affinity, and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, circ-P(EG3Glu)20-IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration in vivo. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.

Salt- and pH-Triggered Helix-Coil Transition of Ionic Polypeptides under Physiology Conditions.

Controlling the helix-coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here, we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EG x)-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P(EG xDMA-Glu) ( x = 1, 2, and 3), show excellent aqueous solubility (>20 mg/mL) regardless of their charge states. Unlike poly-l-lysine that can form a helix only at pH above 10, P(EG xDMA-Glu) undergo a pH-dependent helix-coil switch with their transition points within the physiological range (pH ∼5.3-6.5). Meanwhile, P(EG xDMA-Glu) exhibit an unusual salt-induced helical conformation presumably owing to the unique properties of EG x linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides and represents a facile approach toward stimuli-responsive biopolymers for advanced biological applications.

The annealing helicase HARP protects stalled replication forks.

Mutations in HepA-related protein (HARP) are the only identified causes of Schimke immunoosseous dysplasia (SIOD). HARP has a unique annealing helicase activity in vitro, but the in vivo functional significance remains unknown. Here, we demonstrated that HARP is recruited to stalled replication forks via its direct interaction with Replication protein A (RPA). Cells with HARP depletion displayed increased spontaneous DNA damage and G2/M arrest, suggesting that HARP normally acts to stabilize stalled replication forks. Our data place the annealing helicase activity of HARP at replication forks and propose that SIOD syndrome may be caused by the destabilization of replication forks during cell proliferation.

A Concise Approach to Site-Specific Topological Protein-Poly(amino acid) Conjugates Enabled by in Situ-Generated Functionalities.

Controlling the topology of polymer-modified proteins has attracted growing interest. However, one of the main challenges in this field is the lack of efficient and site-specific methods for installing multiple bioorthogonal functionalities on substrate polymers. We report here an orchestrating strategy that provides easy access to various topological protein-poly(amino acid) (PAA) conjugates in high yields. This method features the in situ installation of two "chemical handles", including a thioester for native chemical ligation and a polyglycine nucleophile for sortase A-mediated ligation, at both ends of substrate PAAs. As a result, neither pre-functionalization of initiator or monomer units, nor post-polymerization modification of the resultant polymers, is necessary. Site-specific topological conjugates, particularly circular conjugates, can be conveniently synthesized under mild conditions from the functionalized PAAs. The biomedical utility of our method is demonstrated by the rapid and efficient generation of several therapeutic interferon-α conjugates, which exhibit significantly enhanced protease resistance and thermostability. Given the versatility of both PAAs and proteins, the method offers a convenient approach to producing libraries of conjugates for biological applications.

Phenyl Trimethylsilyl Sulfide-Mediated Controlled Ring-Opening Polymerization of α-Amino Acid N-Carboxyanhydrides.

We describe here the first example of trimethylsilyl sulfide (S-TMS) mediated controlled ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs). We show that phenyl trimethylsilyl sulfide (PhS-TMS), an inexpensive and commercially available compound, mediates rapid ROP of a broad scope of NCA monomers, produces functional poly(amino acids) (PAAs) with controllable molecular weights (MWs), narrow polydispersity index (PDI), and an in situ generated phenyl thioester group at the C-terminus (PAA-SPhs). PhS-TMS offers more rapid chain initiation than previously reported hexamethyldisilazane (HMDS) initiator, ensuring a living polymerization with better control. Mechanistic studies suggest that a reactive trimethylsilyl carbamate (TMSC) was generated during the chain initiation and continued to regulate the chain propagation through a TMS transfer process. Considering the versatility of NCAs, and the potential of leveraging the C-terminal phenyl thioester for native chemical ligation (NCL), we believe this method may offer a powerful platform enabling the rapid generation of functional PAAs and their C-terminal conjugates for numerous biological applications.

FIGNL1-containing protein complex is required for efficient homologous recombination repair.

The RAD51 recombinase plays a central role in homologous recombination (HR), which is critical for repair of DNA double-strand breaks, maintenance of genomic stability, and prevention of developmental disorders and cancer. Here, we report the identification of an RAD51-binding protein fidgetin-like 1 (FIGNL1). FIGNL1 specifically interacts with RAD51 through its conserved RAD51 binding domain. Cells depleted of FIGNL1 show defective HR repair. Interestingly, FIGNL1 is recruited to sites of DNA damage in a manner that is independent of breast cancer 2, early onset, RAD51, and probably, RAD51 paralogs. Conversely, FIGNL1 depletion does not affect the loading of RAD51 onto ssDNA. Our additional analysis uncovered KIAA0146, also known as scaffolding protein involved in DNA repair (SPIDR), as a binding partner of FIGNL1 and established that KIAA0146/SPIDR acts with FIGNL1 in HR repair. Collectively, our study uncovers a protein complex, which consists of FIGNL1 and KIAA0146/SPIDR, in DNA repair and provides potential directions for cancer diagnosis and therapy.

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Near-infrared active ferrocenyl porous organic polymer with photothermal enhanced enzymatic activity for combination antibacterial application.

As a severe ongoing global problem, bacterial contamination exists in every aspect of human life and the search for new antibacterial agents is urgently needed. Herein, a ferrocenyl porous organic polymer (FMC-POP) broad-spectrum antibacterial agent based on synergistic photothermal and peroxidase-like activity was prepared in a facile manner via the copolymerization of ferrocene diformaldehyde and cinnamaldehyde with mannitol through the acid-responsive acetal bond. The photoactive FMC-POP, with high photothermal conversion efficiency (41.45%), could convert not only the near-infrared laser irradiation into local heat to eradicate bacteria, but also low-concentration H2O2 into radical oxygen species (˙OH) that are effective against bacteria. Compared with single-mode photothermal (PTT) and enzymatic therapies, this combination therapy could significantly improve the bactericidal effect, exhibiting a germicidal efficiency of up to 99% (vs. 80.42% for PTT and 70% for enzyme). Thus, our work paves the way for a synergistic non-invasive antimicrobial therapy, which could expand the applications of POP-based artificial enzymes in biomedicine.

Relatlimab: a novel drug targeting immune checkpoint LAG-3 in melanoma therapy.

Relatlimab is a type of human immunoglobulin G4 monoclonal blocking antibody. It is the world's first Lymphocyte-Activation Gene-3 (LAG-3) inhibitor and the third immune checkpoint inhibitor with clinical application, following PD-1 and CTLA-4. Relatlimab can bind to the LAG-3 receptor which blocks the interaction between LAG-3 and its ligand to reduce LAG-3 pathway-mediated immunosuppression and promote T-cell proliferation, inducing tumor cell death. On 18 March 2022, the U.S. FDA approved the fixed-dose combination of relatlimab developed by Bristol Myers Squibb with nivolumab, under the brand name Opdualag for the treatment of unresectable or metastatic melanoma in adult and pediatric patients aged 12 and older. This study comprehensively describes the mechanism of action and clinical trials of relatlimab and a brief overview of immune checkpoint drugs currently used for the treatment of melanoma.

The role of the human SWI5-MEI5 complex in homologous recombination repair.

The Swi5-Mei5 complex and its homologues are involved in specialized recombination pathways in budding and fission yeasts. Although the fission yeast homologue Swi5-Sfr1 is critical for homologous recombination repair, the budding yeast counterpart Sae3-Mei5 is meiosis-specific, interacts with Dmc1, and promotes assembly of Dmc1 on meiotic chromosomes. Here, we identify and characterize the human SWI5-MEI5 (C9orf119-C10orf78) complex. We showed that SWI5 and MEI5 form a stable complex in vitro and in vivo. The C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 dictate this conserved interaction. In addition, SWI5-MEI5 directly interacts with RAD51 in vitro. Depletion of SWI5 or MEI5 in human cells causes defects in homologous recombination repair. Finally, SWI5- or MEI5-depleted cells display enhanced sensitivity to ionizing radiation, consistent with the role of this complex in HR repair. Our results suggest that human SWI5-MEI5 has an evolutionarily conserved function in homologous recombination repair.

MRE11-RAD50-NBS1 complex dictates DNA repair independent of H2AX.

DNA double-strand breaks (DSBs) represent one of the most serious forms of DNA damage that can occur in the genome. Here, we show that the DSB-induced signaling cascade and homologous recombination (HR)-mediated DSB repair pathway can be genetically separated. We demonstrate that the MRE11-RAD50-NBS1 (MRN) complex acts to promote DNA end resection and the generation of single-stranded DNA, which is critically important for HR repair. These functions of the MRN complex can occur independently of the H2AX-mediated DNA damage signaling cascade, which promotes stable accumulation of other signaling and repair proteins such as 53BP1 and BRCA1 to sites of DNA damage. Nevertheless, mild defects in HR repair are observed in H2AX-deficient cells, suggesting that the H2AX-dependent DNA damage-signaling cascade assists DNA repair. We propose that the MRN complex is responsible for the initial recognition of DSBs and works together with both CtIP and the H2AX-dependent DNA damage-signaling cascade to facilitate repair by HR and regulate DNA damage checkpoints.