N-terminal Research Articles

Conserved molecular recognition by an intrinsically disordered region in the absence of sequence conservation.

Intrinsically disordered regions (IDRs) are critical for cellular function yet often appear to lack sequence conservation when assessed by multiple sequence alignments. This raises the question of if and how function can be encoded and preserved in these regions despite massive sequence variation. To address this question, we have applied coarse-grained molecular dynamics simulations to investigate non-specific RNA binding of coronavirus nucleocapsid proteins. Coronavirus nucleocapsid proteins consist of multiple interspersed disordered and folded domains that bind RNA. Here, we focus on the first two domains of coronavirus nucleocapsid proteins: the disordered N-terminal domain (NTD) and the folded RNA binding domain (RBD). While the NTD is highly variable across evolution, the RBD is structurally conserved. This combination makes the NTD-RBD a convenient model system for exploring the interplay between an IDR adjacent to a folded domain and how changes in IDR sequence can influence molecular recognition of a partner. Our results reveal a surprising degree of sequence-specificity encoded by both the composition and the precise order of the amino acids in the NTD. The presence of an NTD can - depending on the sequence - either suppress or enhance RNA binding. Despite this sensitivity, large-scale variation in NTD sequences is possible while certain sequence features are retained. Consequently, a conformationally-conserved dynamic and disordered RNA:protein complex is found across nucleocapsid protein orthologs despite large-scale changes in both NTD sequence and RBD surface chemistry. Taken together, these insights shed light on the ability of disordered regions to preserve functional characteristics despite their sequence variability.

Aqueous Graphene Dispersion and Biofunctionalization via Enzymatic Oxidation of Tripeptides.

Graphene, a 2D carbon material, possesses extraordinary mechanical, electrical, and thermal properties, making it highly attractive for various biological applications such as biosensing, biotherapeutics, and tissue engineering. However, the tendency of graphene sheets to aggregate and restack hinders its dispersion in water, limiting these applications. Peptides, with their defined amino acid sequences and versatile functionalities, are compelling molecules with which to modify graphene-aromatic amino acids can strengthen interactions through π-stacking and charged groups can be chosen to make the sheets dispersible and stable in water. Here, a facile and green method for covalently functionalizing and dispersing graphene using amphiphilic tripeptides, facilitated by a tyrosine phenol side chain, through an aqueous enzymatic oxidation process is demonstrated. The presence of a second aromatic side chain group enhances this interaction through non-covalent support via π-π stacking with the graphene surface. Futhermore, the addition of charged moieties originating from either ionizable amino acids or terminal groups facilitates profound interactions with water, resulting in the dispersion of the newly functionalized graphene in aqueous solutions. This biofunctionalization method resulted in ≈56% peptide loading on the graphene surface, leading to graphene dispersions that remain stable for months in aqueous solutions outperforming currently used surfactants.

Mapping the membrane orientation of auxin homeostasis regulators PIN5 and PIN8 in Arabidopsis thaliana root cells reveals their divergent topology

PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

A phase 1/2 study of ONCT-534, a dual-action androgen receptor inhibitor (DAARI), in patients with metastatic castration-resistant prostate cancer.

TPS5111 Background: Current therapeutic strategies for metastatic castration-resistant prostate cancer (mCRPC) include treatment with next-generation androgen receptor pathway inhibitors (ARPIs), which target the ligand-binding domain (LBD) of the AR. Approximately 5% to 10% of patients will have primary resistance to ARPIs and most initial responders will develop resistance within 1 to 3 years. Eventually, nearly all men with prostate cancer treated with ARPIs develop resistance via several mechanisms including AR genomic alterations, epigenetic alterations, and expression of truncated constitutively active AR splice variants. Hence, there is substantial unmet need for treatment of men with mCRPC resistant to at least one next-generation ARPI. ONCT-534 is a DAARI with a novel mechanism of action that combines inhibition of AR function with degradation of the AR protein. Importantly, this activity includes interaction with both LBD and the N-terminal domain (NTD) of the AR, rendering it effective against splice variants and LBD mutants, unlike existing ARPIs. ONCT-534 has demonstrated preclinical activity in prostate cancer models against unmutated AR and multiple forms of AR alteration, including amplification, mutations in the LBD, and splice variants with loss of LBD. Methods: ONCT-534-101 is a phase 1/2, multi-center study to evaluate the safety, tolerability, antitumor activity, and pharmacokinetics of ONCT-534 in subjects with histologically confirmed mCRPC without neuroendocrine differentiation or small cell features who have relapsed or are refractory (R/R) to at least one next-generation ARPI. The study is separated into Phase 1 Dose Escalation and a Phase 2 Dose Expansion portions. The Phase 1 portion will evaluate approximately 27 subjects in 5 dose levels using an adaptive Bayesian Optimal Interval (BOIN) design to assess safety, tolerability and dose limiting toxicities (DLTs) at escalating doses and to determine the maximum tolerated dose and inform the 2 dose levels or schedules to be tested in Phase 2. DLTs will be assessed during the first 28 days of treatment. AR phenotype and AR levels will be evaluated for each subject pre- and post-treatment. The Phase 2 portion will evaluate approximately 32 subjects in 2 randomized cohorts to assess safety and tolerability of ONCT-534, compare the 2 different dose levels or schedules to select the optimal dose for further study, and assess the preliminary antitumor activity of ONCT-534. In both phases, after a screening period, eligible subjects with mCRPC will receive their assigned dose regimen of ONCT-534, which will be administered orally daily for 2 years or until disease progression and no longer clinically benefiting from treatment or development of unacceptable toxicity. Cohorts 1 and 2 have been completed without DLT. Enrollment into Cohort 3 began in January 2024 (NCT 05917470). Clinical trial information: NCT05917470 .

PTPN11variants in chronic myelomonocytic leukemia: Phenotypic and prognostic correlates.

6513 Background: PTPN11 (Ch12q24) is a proto-oncogene that codes for SHP2, a regulatory protein tyrosine phosphatase that impacts RAS/MAPK signaling. Germline variants have been described in juvenile myelomonocytic leukemia, while somatic variants have been associated with inferior survival in acute myeloid leukemia. PTPN11 variants are infrequent in chronic myelomonocytic leukemia (CMML), and their impact on disease phenotype and survival remains unclear. Methods: After IRB approval, 396 molecularly annotated CMML patients at Mayo Clinic were screened for somatic PTPN11 variants within 12 months of diagnosis while still in chronic phase disease. Overall (OS), acute myeloid leukemia (AML)-free survival (LFS), and predictors of survival were estimated using standard statistical measures. Results: 396 patients (median age 71 years; males 67%) with CMML were considered; pathogenic PTPN11 variants were seen in 12 (3%) with a median VAF of 20.5% (range: 4-50): A72T (n = 2), F285S (n = 2), and A72S (n = 2). Variants were most likely to be seen in the N-terminal SH2 domain (n = 7), followed by the PTP domain (n = 6), and the C-terminal SH2 domain (n = 1). PTPN11 variants clustered with CMML-2 (p = 0.02), higher absolute neutrophil count (p = 0.04), peripheral blood (p = 0.06) and bone marrow (p = 0.02) blast %, older age (p <0.01), and were less likely to have SRSF2 comutations (p = 0.02). After a median follow-up of 18 months (range 0-193), 253 (64%) deaths and 68 (17%) AML transformations were recorded. PTPN11 variants negatively and independently impacted both OS (median 13 vs. 31 months; p <0.01) and LFS (AML events: 42% (5/12) vs. 16% (63/384); p <0.01). In age-adjusted multivariate analysis restricted to genetic risk factors, survival was negatively affected by abnormal karyotype and mutations involving PTPN11, DNMT3A, and SETBP1, and positively by TET2 mutations. All but SETBP1 retained significance in the presence of additional independent risk factors, namely male sex (p <0.01) and leukocyte count (p <0.01); HR in the latter analysis was 4.3 for PTPN11, 2.8 for DNMT3A, and 1.6 for abnormal karyotype. A similar analysis for LFS identified, as risk factors, PTPN11 (p = 0.01), DNMT3A (p <0.01), and ASXL1 (p = 0.03) mutations, as well as bone marrow blast % (p <0.01), absolute monocyte count ≥10 (p = 0.02), and immature circulating myeloid cells (p <0.01). Conclusions: The current study identifies PTPN11 and DNMT3A mutations as independent risk factors for both OS and LFS in CMML. In regard to additional genetic risk factors, OS was positively affected by TET2 mutations and negatively by abnormal karyotype, while LFS was negatively affected by ASXL1 mutations.

Biochemical and structural characterization of the DNA-binding properties of human TRIP4 ASCH domain reveals insights into its functional role

TRIP4 is a conserved transcriptional coactivator that is involved in the regulation of the expression of multiple genes. It consists of a classical N-terminal C2HC5-like zinc-finger domain and a conserved C-terminal ASCHdomain. Here, we characterized the DNA-binding properties of the human TRIP4 ASCH domain. Our biochemical data show that TRIP4-ASCH has comparable binding affinities toward ssDNA and dsDNA of different lengths, sequences, and structures. The crystal structures reveal that TRIP4-ASCH binds to DNA substrates in a sequence-independent manner through two adjacent positively charged surface patches: one binds to the 5'-end of DNA, and the other binds to the 3'-end of DNA. Further mutagenesis experiments and binding assays confirm the functional roles of key residues involved in DNA binding. In summary, our data demonstrate that TRIP4-ASCH binds to the 5' and 3'-ends of DNA in a sequence-independent manner, which will facilitate further studies of the biological function of TRIP4.

The activation of Chlamydomonas reinhardtii alpha amylase 2 by glutamine requires its N-terminal aspartate kinase-chorismate mutase-tyrA (ACT) domain.

The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of Chlamydomonas reinhardtii that we captured by a cyclic guanosine monophosphate (cGMP) affinity chromatography approach, namely, soluble starch synthase STA3, starch-branching enzyme SBE1, and α-amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzyme's N-terminal aspartate kinase-chorismate mutase-tyrA domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N and C metabolism at the enzymatic level, increasing the liberation of C skeletons from starch when high Gln levels signal an abundance of assimilated N.

RASEF/Rab45 regulates the formation and sorting of zymogen granules and secretion of digestive enzymes by pancreatic acinar cells

The pancreas is a glandular organ with both endocrine and exocrine functions. Researchers have investigated the roles of several Rab proteins, which are major regulators of membrane trafficking, in pancreatic exocytosis of zymogen granules in exocrine cells, also known as acinar cells. However, detailed molecular mechanisms mediated by Rab proteins are not fully understood. RASEF/Rab45 is an atypical Rab GTPase that contains N-terminal EF-hand and coiled-coil domains, as well as a C-terminal Rab-GTPase domain. In this study, we investigated the in vivo role of RASEF in pancreatic acinar cells using RASEF-knockout (KO) mice. Morphological analyses revealed that pancreatic acinar cells in RASEF-KO mice had an increased number of zymogen granules and abnormal formations of organelles, such as the endoplasmic reticulum (ER) and lysosomes. Biochemical analyses showed that ER proteins were decreased, but digestive enzymes were increased in the RASEF-KO pancreas. Moreover, trypsinogen was activated and co-localized with the endo-lysosomal marker LAMP1 in RASEF-KO pancreas. Upon cerulein administration to induce acute pancreatitis, impaired enzyme release from the pancreas was observed in the serum of RASEF-KO mice. These findings suggest that RASEF likely regulates the formation and sorting of zymogen granules and secretion of digestive enzymes by pancreatic acinar cells.

Human Coronavirus 229E Infection Inactivates Pyroptosis Executioner Gasdermin D but Ultimately Leads to Lytic Cell Death Partly Mediated by Gasdermin E.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

Mechanism of heterochromatin remodeling revealed by the DDM1 bound nucleosome structures

The SWI/SNF2 chromatin remodeling factor decreased DNA methylation 1 (DDM1) is essential for the silencing of transposable elements (TEs) in both euchromatic and heterochromatic regions. Here, we determined the cryo-EM structures of DDM1-nucleosomeH2A and DDM1-nucleosomeH2A.W complexes at near-atomic resolution in the presence of the ATP analog ADP-BeFx. The structures show that nucleosomal DNA is unwrapped more on the surface of the histone octamer containing histone H2A than that containing histone H2A.W. DDM1 embraces one DNA gyre of the nucleosome and interacts with the N-terminal tails of histone H4. Although we did not observe DDM1-H2A.W interactions in our structures, the results of the pull-down experiments suggest a direct interaction between DDM1 and the core region of histone H2A.W. Our work provides mechanistic insights into the heterochromatin remodeling process driven by DDM1 in plants.

Androgen Signaling in Prostate Cancer: When a Friend Turns Foe.

Androgen (AR) signaling is the main signaling for the development of the prostate and its normal functioning. AR is highly specific for testosterone and dihydrotestosterone, significantly contributing to prostate development, physiology, and cancer. All these receptors have emerged as crucial therapeutic targets for PCa. In the year 1966, the Noble prize was awarded to Huggins and Hodge for their groundbreaking discovery of AR. As it is a pioneer transcription factor, it belongs to the steroid hormone receptor family and consists of domains, including DNA binding domain (DBD), hormone response elements (HRE), C-terminal ligand binding domain (LBD), and N-terminal regulatory domains. Structural variations in AR, such as AR gene amplification, LBD mutations, alternative splicing of exons, hypermethylation of AR, and co- regulators, are major contributors to PCa. It's signaling is crucial for the development and functioning of the prostate gland, with the AR being the key player. The specificity of AR for testosterone and dihydrotestosterone is important in prostate physiology. However, when it is dysregulated, AR contributes significantly to PCa. However, the structural variations in AR, such as gene amplification, mutations, alternative splicing, and epigenetic modifications, drive the PCa progression. Therefore, understanding AR function and dysregulation is essential for developing effective therapeutic strategies. Thus, the aim of this review was to examine how AR was initially pivotal for prostate development and how it turned out to show both positive and detrimental implications for the prostate.

The intrinsically disordered transactivation region of HOXA9 regulates its function by auto-inhibition of its DNA-binding activity

HOXA9 transcription factor is expressed in hematopoietic stem cells and is involved in the regulation of their differentiation and maturation to various blood cells. HOXA9 is linked to various leukemia and is a marker for poor prognosis of acute myeloid leukemia (AML). This protein has a conserved DNA-binding homeodomain and a transactivation domain. We show that this N-terminal transactivation domain is intrinsically disordered and inhibits DNA-binding by the homeodomain. Using NMR spectroscopy and molecular dynamics simulation, we show that the hexapeptide 197AANWLH202 in the disordered region transiently occludes the DNA-binding interface. The hexapeptide also forms a rigid segment, as determined by NMR dynamics, in an otherwise flexible disordered region. Interestingly, this hexapeptide is known to mediate the interaction of HOXA9 and its TALE partner proteins, such as PBX1, and help in cooperative DNA binding. Mutation of tryptophan to alanine in the hexapeptide abrogates the DNA-binding auto-inhibition. We propose that the disordered transactivation region plays a dual role in the regulation of HOXA9 function. In the absence of TALE partners, it inhibits DNA binding, and in the presence of TALE partners it interacts with the TALE protein and facilitates the cooperative DNA binding by the HOX-TALE complex.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

An N-terminal acidic β-sheet domain is responsible for the metal-accumulation properties of amyloid-β protofibrils: a molecular dynamics study

The influence of metal ions on the structure of amyloid-β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta $$\\end{document} (Aβ) protofibril models was studied through molecular dynamics to explore the molecular mechanisms underlying metal-induced Aβ aggregation relevant in Alzheimer’s disease (AD). The models included 36-, 48-, and 188-mers of the Aβ42 sequence and two disease-modifying variants. Primary structural effects were observed at the N-terminal domain, as it became susceptible to the presence of cations. Specially when β-sheets predominate, this motif orients N-terminal acidic residues toward one single face of the β-sheet, resulting in the formation of an acidic region that attracts cations from the media and promotes the folding of the N-terminal region, with implications in amyloid aggregation. The molecular phenotype of the protofibril models based on Aβ variants shows that the AD-causative D7N mutation promotes the formation of N-terminal β-sheets and accumulates more Zn2+, in contrast to the non-amyloidogenic rodent sequence that hinders the β-sheets and is more selective for Na+ over Zn2+ cations. It is proposed that forming an acidic β-sheet domain and accumulating cations is a plausible molecular mechanism connecting the elevated affinity and concentration of metals in Aβ fibrils to their high content of β-sheet structure at the N-terminal sequence.Graphic abstract

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

New insights into the domain of unknown function (DUF) of EccC5, the pivotal ATPase providing the secretion driving force to the ESX-5 secretion system.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

Golm1 facilitates the CaO2-DOPC-DSPE200-PEI -CsPbBr3 QDs -induced apoptotic death of hepatocytes through the stimulation of mitochondrial autophagy and mitochondrial reactive oxygen species production through interactions with P53/Beclin-1/Bcl-2

Mitophagy is a distinct physiological process that can have beneficial or deleterious effects in particular tissues. Prior research suggests that mitophagic activity can be triggered by CaO2-PM-CsPbBr3 QDs, yet the specific role that mitophagy plays in hepatic injury induced by CaO2-PM-CsPbBr3 QDs has yet to be established. Accordingly, in this study a series of mouse model- and cell-based experiments were performed that revealed the ability of CaO2-PM-CsPbBr3 QDs to activate mitophagic activity. Golm1 was upregulated in response to CaO2-PM-CsPbBr3 QDs treatment, and overexpressing Golm1 induced autophagic flux in the murine liver and hepatocytes, whereas knocking down Golm1 had the opposite effect. CaO2-PM-CsPbBr3 QDs were also able to Golm1 expression, in turn promoting the degradation of P53 and decreasing the half-life of this protein. Overexpressing Golm1 was sufficient to suppress the apoptotic death of hepatocytes in vitro and in vivo, whereas the knockdown of Golm1 had the opposite effect. The ability of Golm1 to promote p53-mediated autophagy was found to be associated with the disruption of Beclin-1 binding to Bcl-2, and the Golm1 N-terminal domain was determined to be required for p53 interactions, inducing autophagic activity in a manner independent of helicase activity or RNA binding. Together, these results indicate that inhibiting Golm1 can promote p53-dependent autophagy via disrupting Beclin-1 binding to Bcl-2, highlighting a novel approach to mitigating liver injury induced by CaO2-PM-CsPbBr3 QDs.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.