Pathogenic Proteins Research Articles

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Vaccine enhancement and improved immunogenicity using erythrocytes as carriers

Achieving optimal immunogenicity and efficacy in vaccination presents significant challenges, including the need for multiple doses and the limited immunogenicity observed with certain vaccine formulations. To overcome these obstacles, researchers are exploring supplementary components and innovative delivery strategies. The development of an effective vaccine delivery technology is crucial for achieving sufficient protective immunity and disease prevention. This article focuses on the potential of erythrocytes as vaccine carriers to enhance vaccine effectiveness, particularly in the context of whole particle vaccines. The extended lifespan of erythrocytes enables sustained antigen exposure, resulting in continuous antigen presentation to immune cells. Moreover, erythrocytes exhibit biocompatibility, a well-established safety profile, and the capacity to modulate immune responses through interactions with complement receptors. These characteristics position erythrocytes as an appealing platform for targeted vaccine delivery. The hypothesis suggests utilizing bispecific monoclonal antibodies to complement receptor 1 and specific pathogen proteins to hitchhike inactivated pathogens onto erythrocytes. These modified erythrocytes can then serve as carriers to deliver the whole particle vaccine to antigen-presenting cells (APCs) or function as APCs themselves in the spleen. This article puts forward treatment protocols aimed at improving accessibility of vaccinations at the appropriate location, reducing the frequency of vaccine doses, and stimulating sufficient protective immunity.

Effector MoSDT1 enhances Magnaporthe oryzae virulence and plays a dual role in regulating rice defense.

C2H2 zinc effectors are a class of pathogen proteins that play a dual role in plant-pathogen interactions, promoting pathogenicity and enhancing plant defense. In our previous research, we identified Magnaporthe oryzae Systemic Defense Trigger 1 (MoSDT1) as a C2H2 zinc effector that activates rice (Oryza sativa) defense when overexpressed in rice. However, its regulatory roles in pathogenicity and defense require further investigation. In this study, we generated an MoSDT1 overexpressing strain and 2 knockout strains of M. oryzae to assess the impact of MoSDT1 on pathogenicity, rice defense, and phenotypic characteristics. Our analyses revealed that MoSDT1 substantially influenced vegetative growth, conidia size, and conidiation, and was crucial for the virulence of M. oryzae while suppressing rice defense. MoSDT1 localized to the nucleus and cytoplasm of rice, either dependent or independent of M. oryzae delivery. Through RNA-seq, scRNA-seq, and ChIP-seq, we identified that MoSDT1 modulates rice defense by regulating the phosphorylation and ubiquitination of various rice signaling proteins, including transcription factors, transcription repressors, kinases, phosphatases, and the ubiquitin system. These findings provide valuable insights into the regulatory mechanisms of C2H2 zinc finger effector proteins and offer important foundational information for utilizing their target genes in disease resistance breeding and the design of targets for disease management.

Extraneural infection route restricts prion conformational variability and attenuates the impact of quaternary structure on infectivity.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Annexin A2: A Double-Edged Sword in Pathogen Infection.

Annexin A2 (ANXA2) is a multifunctional calcium- and phospholipid-binding protein that plays an important role in various cells. During pathogen infections, ANXA2 modulates the nuclear factor kappa-B (NF-κB) and cell apoptosis signaling pathways and guides the chemotaxis of inflammatory cells toward inflammation sites, thereby protecting the host organism through the modulation of the inflammatory response. In addition, ANXA2 can regulate immune responses, and in certain pathogen infections, it can interact with pathogen proteins to facilitate their invasion and proliferation. This review provides an overview of the research progress on how ANXA2 regulates pathogen infections.

Definition of the effector landscape across 13 phytoplasma proteomes with LEAPH and EffectorComb.

'Candidatus Phytoplasma' genus, a group of fastidious phloem-restricted bacteria, can infect a wide variety of both ornamental and agro-economically important plants. Phytoplasmas secrete effector proteins responsible for the symptoms associated with the disease. Identifying and characterizing these proteins is of prime importance for expanding our knowledge of the molecular bases of the disease. We faced the challenge of identifying phytoplasma's effectors by developing LEAPH, a machine learning ensemble predictor composed of four models. LEAPH was trained on 479 proteins from 53 phytoplasma species, described by 30 features. LEAPH achieved 97.49% accuracy, 95.26% precision and 98.37% recall, ensuring a low false-positive rate and outperforming available state-of-the-art methods. The application of LEAPH to 13 phytoplasma proteomes yields a comprehensive landscape of 2089 putative pathogenicity proteins. We identified three classes according to different secretion models: 'classical', 'classical-like' and 'non-classical'. Importantly, LEAPH identified 15 out of 17 known experimentally validated effectors belonging to the three classes. Furthermore, to help the selection of novel candidates for biological validation, we applied the Self-Organizing Maps algorithm and developed a Shiny app called EffectorComb. LEAPH and the EffectorComb app can be used to boost the characterization of putative effectors at both computational and experimental levels, and can be employed in other phytopathological models.

Liquid-liquid phase separation in diseases.

Liquid-liquid phase separation (LLPS), an emerging biophysical phenomenon, can sequester molecules to implement physiological and pathological functions. LLPS implements the assembly of numerous membraneless chambers, including stress granules and P-bodies, containing RNA and protein. RNA-RNA and RNA-protein interactions play a critical role in LLPS. Scaffolding proteins, through multivalent interactions and external factors, support protein-RNA interaction networks to form condensates involved in a variety of diseases, particularly neurodegenerative diseases and cancer. Modulating LLPS phenomenon in multiple pathogenic proteins for the treatment of neurodegenerative diseases and cancer could present a promising direction, though recent advances in this area are limited. Here, we summarize in detail the complexity of LLPS in constructing signaling pathways and highlight the role of LLPS in neurodegenerative diseases and cancers. We also explore RNA modifications on LLPS to alter diseases progression because these modifications can influence LLPS of certain proteins or the formation of stress granules, and discuss the possibility of proper manipulation of LLPS process to restore cellular homeostasis or develop therapeutic drugs for the eradication of diseases. This review attempts to discuss potential therapeutic opportunities by elaborating on the connection between LLPS, RNA modification, and their roles in diseases.

Sesquiterpene coumarins of Asafoetida mask the functional site of pathogenic proteins of SARS-CoV-2 to combat COVID-19 disease

Sesquiterpene coumarins of Asafoetida mask the functional site of pathogenic proteins of SARS-CoV-2 to combat COVID-19 disease

Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.

Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.

Unimolecular Chemiexcited Oxygenation of Pathogenic Amyloids

AbstractPathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid‐β (Aβ), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA‐Cl and dmCLA‐Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA‐Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA‐Cl‐acid. dmCLA‐Cl‐acid promoted oxygenation of Aβ amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.

Unimolecular Chemiexcited Oxygenation of Pathogenic Amyloids.

Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-β (Aβ), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aβ amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.

Rab27b promotes lysosomal function and alpha-synuclein clearance in neurons.

Alpha-synuclein (αsyn) is the key pathogenic protein implicated in synucleinopathies including Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB). In these diseases, αsyn is thought to spread between cells where it accumulates and induces pathology; however, mechanisms that drive its propagation or aggregation are poorly understood. We have previously reported that the small GTPase Rab27b is elevated in human PD and DLB and that it can mediate the autophagic clearance and toxicity of αsyn in a paracrine αsyn cell culture neuronal model. Here, we expanded our previous work and further characterized a role for Rab27b in neuronal lysosomal processing and αsyn clearance. We found that Rab27b KD in this αsyn inducible neuronal model resulted in lysosomal dysfunction and increased αsyn levels in lysosomes. Similar lysosomal proteolytic defects and enzymatic dysfunction were observed in both primary neuronal cultures and brain lysates from Rab27b knockout (KO) mice. αSyn aggregation was exacerbated in Rab27b KO neurons upon treatment with αsyn preformed fibrils. We found no changes in lysosomal counts or lysosomal pH in either model, but we did identify defects in acidic vesicle trafficking in Rab27b KO primary neurons which may drive lysosomal dysfunction and promote αsyn aggregation. Rab27b OE enhanced lysosomal activity and reduced insoluble αsyn accumulation. Finally we found elevated Rab27b levels in human postmortem incidental Lewy Body Disease (iLBD) subjects relative to healthy controls. These data suggest a role for Rab27b in neuronal lysosomal activity and identify it as a potential therapeutic target in synucleinopathies.

Supramolecular artificial Nano-AUTACs enable tumor-specific metabolism protein degradation for synergistic immunotherapy.

Autophagy-targeting chimera (AUTAC) has emerged as a powerful modality that can selectively degrade tumor-related pathogenic proteins, but its low bioavailability and nonspecific distribution significantly restrict their therapeutic efficacy. Inspired by the guanine structure of AUTAC molecules, we here report supramolecular artificial Nano-AUTACs (GM NPs) engineered by AUTAC molecule GN [an indoleamine 2,3-dioxygenase (IDO) degrader] and nucleoside analog methotrexate (MTX) through supramolecular interactions for tumor-specific protein degradation. Their nanostructures allow for precise localization and delivery into cancer cells, where the intracellular acidic environment can disrupt the supramolecular interactions to release MTX for eradicating tumor cells, modulating tumor-associated macrophages, activating dendritic cells, and inducing autophagy. Specifically, the induced autophagy facilitates the released GN for degrading immunosuppressive IDO to further enhance effector T cell activity and inhibit tumor growth and metastasis. This study offers a unique strategy for building a nanoplatform to advance the field of AUTAC in tumor immunotherapy.

Research progress on astrocyte-derived extracellular vesicles in the pathogenesis and treatment of neurodegenerative diseases.

Neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), pose significant global health risks and represent a substantial public health concern in the contemporary era. A primary factor in the pathophysiology of these disorders is aberrant accumulation and aggregation of pathogenic proteins within the brain and spinal cord. Recent investigations have identified extracellular vesicles (EVs) in the central nervous system (CNS) as potential carriers for intercellular transport of misfolded proteins associated with neurodegenerative diseases. EVs are involved in pathological processes that contribute to various brain disorders including neurodegenerative disorders. Proteins linked to neurodegenerative disorders are secreted and distributed from cell to cell via EVs, serving as a mechanism for direct intercellular communication through the transfer of biomolecules. Astrocytes, as active participants in CNS intercellular communication, release astrocyte-derived extracellular vesicles (ADEVs) that are capable of interacting with diverse target cells. This review primarily focuses on the involvement of ADEVs in the development of neurological disorders and explores their potential dual roles- both advantageous and disadvantageousin the context of neurological disorders. Furthermore, this review examines the current studies investigating ADEVs as potential biomarkers for the diagnosis and treatment of neurodegenerative diseases. The prospects and challenges associated with the application of ADEVs in clinical settings were also comprehensively reviewed.

First Report of Single Nucleotide Polymorphisms (SNPs) of the Leporine Shadow of Prion Protein Gene (SPRN) and Absence of Nonsynonymous SNPs in the Open Reading Frame (ORF) in Rabbits.

Prion disorders are fatal infectious diseases that are caused by a buildup of pathogenic prion protein (PrPSc) in susceptible mammals. According to new findings, the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) is associated with prion protein (PrP), promoting the progression of prion diseases. Although genetic polymorphisms in SPRN are associated with susceptibility to several prion diseases, genetic polymorphisms in the rabbit SPRN gene have not been investigated in depth. We discovered two novel single nucleotide polymorphisms (SNPs) in the leporine SPRN gene on chromosome 18 and found strong linkage disequilibrium (LD) between them. Additionally, strong LD was not found between the polymorphisms of PRNP and SPRN genes in rabbits. Furthermore, nonsynonymous SNPs that alter the amino acid sequences within the open reading frame (ORF) of SPRN have been observed in prion disease-susceptible animals, but this is the first report in rabbits. As far as we are aware, this study represents the first examination of the genetic features of the rabbit SPRN gene.

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hhtex1) with a 35 polyglutamine repeat, httex1Q35, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of httex1Q35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1H-15N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" httex1Q35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, httex1Q7, lend confidence to the conclusions drawn from the fits to the httex1Q35 aggregation kinetics.

Mechanical force of uterine occupation enables large vesicle extrusion from proteostressed maternal neurons.

Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.

Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant.

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.