Palmitoylation is required for Sept8-204 and Sept5 to form vesicle-like structure and colocalize with synaptophysin.

Sept8 is a vesicle associated protein and there are two typical transcriptional variants (Sept8-204 and Sept8-201) expressed in mice brain. Interestingly, the coexpression of Sept8-204/Sept5 induces the formation of small sized vesicle-like structure, while that of the Sept8-201/Sept5 produces large puncta. Sept8 is previously shown to be palmitoylated. Here it was further revealed that protein palmitoylation is required for Sept8-204/Sept5 to maintain small sized vesicle-like structure and colocalize with synaptophysin, since either the expression of nonpalmitoylated Sept8-204 mutant (Sept8-204-3CA) or inhibiting Sept8-204 palmitoylation by 2-BP with Sept5 produces large puncta, which barely colocalizes with synaptophysin (SYP). Moreover, it was shown that the dynamic palmitoylation of Sept8-204 is controlled by ZDHHC17 and PPT1, loss of ZDHHC17 decreases Sept8-204 palmitoylation and induces large puncta, while loss of PPT1 increases Sept8-204 palmitoylation and induces small sized vesicle-like structure. Together, these findings suggest that palmitoylation is essential for the maintenance of the small sized vesicle-like structure for Sept8-204/Sept5, and may hint their important roles in synaptic functions.

GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice.

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.

Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin.

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.

Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants.

The constant emergence of SARS-CoV-2 variants continues to impair the efficacy of existing neutralizing antibodies, especially XBB.1.5 and EG.5, which showed exceptional immune evasion properties. Here, we identify a highly conserved neutralizing epitope targeted by a broad-spectrum neutralizing antibody BA7535, which demonstrates high neutralization potency against not only previous variants, such as Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5, but also more recently emerged Omicron subvariants, including BF.7, CH.1.1, XBB.1, XBB.1.5, XBB.1.9.1, EG.5. Structural analysis of the Omicron Spike trimer with BA7535-Fab using cryo-EM indicates that BA7535 recognizes a highly conserved cryptic receptor-binding domain (RBD) epitope, avoiding most of the mutational hot spots in RBD. Furthermore, structural simulation based on the interaction of BA7535-Fab/RBD complexes dissects the broadly neutralizing effect of BA7535 against latest variants. Therapeutic and prophylactic treatment with BA7535 alone or in combination with BA7208 protected female mice from the circulating Omicron BA.5 and XBB.1 variant infection, suggesting the highly conserved neutralizing epitope serves as a potential target for developing highly potent therapeutic antibodies and vaccines.

A copper-responsive global repressor regulates expression of diverse membrane-associated transporters and bacterial drug resistance in mycobacteria.

Sequencing of entire bacterial genomes has led to the identification of many membrane-associated transporters, including several multidrug resistance transport proteins, in recent years. However, the regulators and signaling pathways involved in the expression of these genes remain largely unknown. In this study, we have identified Ms2173, a GntR/FadR family transcription factor, as a novel global regulator in Mycobacterium smegmatis. Ms2173 was found to specifically recognize a 15-bp palindromic motif and to broadly regulate expression of 292 genes, including 37 genes that encode membrane-associated transport proteins. Copper ions induced Ms2173 to form inactive proteins lacking DNA-binding activity. Ms2173 was shown to function as a repressor of its target genes. Interestingly, we found that the function of Ms2173 was linked to mycobacterial drug resistance. Compared with the substantially enhanced drug resistance in the Ms2173-deleted mutant strain, the strains overexpressing Ms2173 were more sensitive to anti-tuberculosis drugs than the wild-type strain. Additionally, copper ions could partially counteract the in vivo function of Ms2173. We have thus characterized the first mycobacterial GntR/Fad-like transcription factor that functions as a copper ion-responsive global repressor that we have renamed GfcR. These findings further enhance our understanding of membrane-associated transporter regulation and drug resistance in mycobacteria.

Palmitoylated Sept8-204 modulates learning and anxiety by regulating filopodia arborization and actin dynamics.

Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.

Biparatopic antibody BA7208/7125 effectively neutralizes SARS-CoV-2 variants including Omicron BA.1-BA.5.

SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1-BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.

A proteome-scale identification of novel antigenic proteins in Mycobacterium tuberculosis toward diagnostic and vaccine development.

Tuberculosis (TB) remains to be a major infectious disease throughout the world. However, the current vaccine for TB has variable protective efficacy, and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use. One of the potential strategies in developing a new diagnostic method and in improving the TB vaccine involves the identification of novel antigenic candidates. This paper aims to identify systematically the novel antigenic proteins with the greatest potential as protective or diagnostic antigens by using the differential response of Mycobacterium tuberculosis proteins to serum from TB patients and healthy individuals. Approximately 87% of the open reading frames of M. tuberculosis were successfully cloned into IPTG-inducible expression vectors. The clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for antigenicity using a mixture of sera from 15 TB patients. Out of the 3480 proteins screened, 249 proteins had significant reactions with the serum samples. Among the 249 proteins, 20 proteins were identified as most reactive. Compared with the commercial test kits, 3 novel antigens from the top 20 proteins, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy. These newly identified antigenic proteins may be used as candidates for serodiagnostic application and vaccine development. Overall, this study's findings may serve as an essential reference for developing new TB diagnostic methods and more effective tuberculosis vaccines.

Identifying the Potential Substrates of the Depalmitoylation Enzyme Acyl-protein Thioesterase 1.

BACKGROUND: The homeostasis of palmitoylation and depalmitoylation is involved in various cellular processes, the disruption of which induces severe physiological consequences. Acyl-protein thioesterase (APT) and palmitoyl-protein thioesterases (PPT) catalyze the depalmitoylation process. The natural mutation in human PPT1 caused neurodegenerative disease, yet the understanding of APT1 remains to be elucidated. While the deletion of APT1 in mice turned out to be potentially embryonically lethal, the decoding of its function strictly relied on the identification of its substrates. OBJECTIVE: To determine the potential substrates of APT1 by using the generated human APT1 knockout cell line. METHODS: The combined techniques of palmitoyl-protein enrichment and massspectrometry were used to analyze the different proteins. Palmitoyl-proteins both in HEK293T and APT1-KO cells were extracted by resin-assisted capture (RAC) and data independent acquisition (DIA) quantitative method of proteomics for data collection. RESULTS: In total, 382 proteins were identified. The gene ontology classification segregated these proteins into diverse biological pathways e.g. endoplasmic reticulum process and ubiquitin-mediated proteolysis. A few potential substrates were selected for verification; indeed, major proteins were palmitoylated. Importantly, their levels of palmitoylation were clearly changed in APT1-KO cells. Interestingly, the proliferation of APT1-KO cells escalated dramatically as compared to that of the WT cells, which could be rescued by APT1 overexpression. CONCLUSION: Our study provides a large scale of potential substrates of APT1, thus facilitating the understanding of its intervened molecular functions.