DNALI1 Promotes Neurodegeneration after Traumatic Brain Injury via Inhibition of Autophagosome-Lysosome Fusion.

Traumatic brain injury (TBI) leads to progressive neurodegeneration that may be caused by chronic traumatic encephalopathy (CTE). However, the precise mechanism remains unclear. Herein, the study identifies a crucial protein, axonemal dynein light intermediate polypeptide 1 (DNALI1), and elucidated its potential pathogenic role in post-TBI neurodegeneration. The DNALI1 gene is systematically screened through analyses of Aging, Dementia, and TBI studies, confirming its elevated expression both in vitro and in vivo. Moreover, it is observed that altered DNALI1 expression under normal conditions has no discernible effect. However, upon overexpression, DNALI1 inhibits autophagosome-lysosome fusion, reduces autophagic flux, and exacerbates cell death under pathological conditions. DNALI1 silencing significantly enhances autophagic flux and alleviates neurodegeneration in a CTE model. These findings highlight DNALI1 as a potential key target for preventing TBI-related neurodegeneration.

Age-, sex- and proximal-distal-resolved multi-omics identifies regulators of intestinal aging in non-human primates.

The incidence of intestinal diseases increases with age, yet the mechanisms governing gut aging and its link to diseases, such as colorectal cancer (CRC), remain elusive. In this study, while considering age, sex and proximal-distal variations, we used a multi-omics approach in non-human primates (Macaca fascicularis) to shed light on the heterogeneity of intestinal aging and identify potential regulators of gut aging. We explored the roles of several regulators, including those from tryptophan metabolism, in intestinal function and lifespan in Caenorhabditis elegans. Suggesting conservation of region specificity, tryptophan metabolism via the kynurenine and serotonin (5-HT) pathways varied between the proximal and distal colon, and, using a mouse colitis model, we observed that distal colitis was more sensitive to 5-HT treatment. Additionally, using proteomics analysis of human CRC samples, we identified links between gut aging and CRC, with high HPX levels predicting poor prognosis in older patients with CRC. Together, this work provides potential targets for preventing gut aging and associated diseases.

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.

The crystal structure of human ferroptosis suppressive protein 1 in complex with flavin adenine dinucleotide and nicotinamide adenine nucleotide.

Ferroptosis is a recently discovered form of regulated cell death characterized by its distinct dependence on iron and the peroxidation of lipids within cellular membranes. Ferroptosis plays a crucial role in physiological and pathological situations and has attracted the attention of numerous scientists. Ferroptosis suppressive protein 1 (FSP1) is one of the main regulators that negatively regulates ferroptosis through the GPX4-independent FSP1-CoQ10-NAD(P)H axis and is a potential therapeutic target for ferroptosis-related diseases. However, the crystal structure of FSP1 has not been resolved, which hinders the development of therapeutic strategies targeting FSP1. To unravel this puzzle, we purified the human FSP1 (hFSP1) protein using the baculovirus eukaryotic cell expression system and solved its crystal structure at a resolution of 1.75 Å. Furthermore, we evaluated the oxidoreductase activity of hFSP1 with NADH as the substrate and identified E156 as the key amino acid in maintaining hFSP1 activity. Interestingly, our results indicated that hFSP1 exists and functions in a monomeric state. Mutagenesis analysis revealed the critical role of the C-terminal domain in the binding of substrate. These findings significantly enhance our understanding of the functional mechanism of FSP1 and provide a precise model for further drug development.

The C9orf72-SMCR8 complex suppresses primary ciliogenesis as a RAB8A GAP.

Approximately half of the familial cases of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD) are attributed to the abnormal GGGGCC repeat expansion within the first intron of C9orf72, potentializing C9orf72 and its product as the most promising target for ALS therapeutics. Nevertheless, the biological function of C9orf72 remains unclear. Previously, we reported that C9orf72 and its binding partner, SMCR8, form a GTPase-activating protein (GAP) complex, which is proposed to regulate membrane trafficking and autophagy. Hereby, we found that the C9orf72-SMCR8 complex negatively regulates primary ciliogenesis and hedgehog (HH) signaling. Furthermore, the biochemical analysis and cell biology experiments identified C9orf72 as the RAB8A binding subunit and SMCR8 as the GAP subunit within the complex. Further, we discussed the relationship among the C9orf72-SMCR8 complex, primary ciliogenesis, and autophagy.

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.

ALS-linked C9orf72-SMCR8 complex is a negative regulator of primary ciliogenesis.

Massive GGGGCC (G4C2) repeat expansion in C9orf72 and the resulting loss of C9orf72 function are the key features of ~50% of inherited amyotrophic lateral sclerosis and frontotemporal dementia cases. However, the biological function of C9orf72 remains unclear. We previously found that C9orf72 can form a stable GTPase activating protein (GAP) complex with SMCR8 (Smith-Magenis chromosome region 8). Herein, we report that the C9orf72-SMCR8 complex is a major negative regulator of primary ciliogenesis, abnormalities in which lead to ciliopathies. Mechanistically, the C9orf72-SMCR8 complex suppresses the primary cilium as a RAB8A GAP. Moreover, based on biochemical analysis, we found that C9orf72 is the RAB8A binding subunit and that SMCR8 is the GAP subunit in the complex. We further found that the C9orf72-SMCR8 complex suppressed the primary cilium in multiple tissues from mice, including but not limited to the brain, kidney, and spleen. Importantly, cells with C9orf72 or SMCR8 knocked out were more sensitive to hedgehog signaling. These results reveal the unexpected impact of C9orf72 on primary ciliogenesis and elucidate the pathogenesis of diseases caused by the loss of C9orf72 function.

The mechanism of ferroptosis and its related diseases.

Ferroptosis, a regulated form of cellular death characterized by the iron-mediated accumulation of lipid peroxides, provides a novel avenue for delving into the intersection of cellular metabolism, oxidative stress, and disease pathology. We have witnessed a mounting fascination with ferroptosis, attributed to its pivotal roles across diverse physiological and pathological conditions including developmental processes, metabolic dynamics, oncogenic pathways, neurodegenerative cascades, and traumatic tissue injuries. By unraveling the intricate underpinnings of the molecular machinery, pivotal contributors, intricate signaling conduits, and regulatory networks governing ferroptosis, researchers aim to bridge the gap between the intricacies of this unique mode of cellular death and its multifaceted implications for health and disease. In light of the rapidly advancing landscape of ferroptosis research, we present a comprehensive review aiming at the extensive implications of ferroptosis in the origins and progress of human diseases. This review concludes with a careful analysis of potential treatment approaches carefully designed to either inhibit or promote ferroptosis. Additionally, we have succinctly summarized the potential therapeutic targets and compounds that hold promise in targeting ferroptosis within various diseases. This pivotal facet underscores the burgeoning possibilities for manipulating ferroptosis as a therapeutic strategy. In summary, this review enriched the insights of both investigators and practitioners, while fostering an elevated comprehension of ferroptosis and its latent translational utilities. By revealing the basic processes and investigating treatment possibilities, this review provides a crucial resource for scientists and medical practitioners, aiding in a deep understanding of ferroptosis and its effects in various disease situations.

DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.

In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.

Autophagy is regulated by endoplasmic reticulum calcium homeostasis and sphingolipid metabolism.

Calcium is involved in a variety of cellular processes. As the crucial components of cell membranes, sphingolipids also play important roles as signaling molecules. Intracellular calcium homeostasis, autophagy initiation and sphingolipid synthesis are associated with the endoplasmic reticulum (ER). Recently, through genetic screening and lipidomics analysis in Saccharomyces cerevisiae, we found that the ER calcium channel Csg2 converts sphingolipid metabolism into macroautophagy/autophagy regulation by controlling ER calcium homeostasis. The results showed that Csg2 acts as a calcium channel to mediate ER calcium efflux into the cytoplasm, and deletion of CSG2 causes a distinct increase of ER calcium concentration, thereby disrupting the stability of the sphingolipid synthase Aur1, leading to the accumulation of the bioactive sphingolipid phytosphingosine (PHS), which specifically and completely blocks autophagy. In summary, our work links calcium homeostasis, sphingolipid metabolism, and autophagy initiation via the ER calcium channel Csg2.

Cellular mechanotransduction in health and diseases: from molecular mechanism to therapeutic targets.

Cellular mechanotransduction, a critical regulator of numerous biological processes, is the conversion from mechanical signals to biochemical signals regarding cell activities and metabolism. Typical mechanical cues in organisms include hydrostatic pressure, fluid shear stress, tensile force, extracellular matrix stiffness or tissue elasticity, and extracellular fluid viscosity. Mechanotransduction has been expected to trigger multiple biological processes, such as embryonic development, tissue repair and regeneration. However, prolonged excessive mechanical stimulation can result in pathological processes, such as multi-organ fibrosis, tumorigenesis, and cancer immunotherapy resistance. Although the associations between mechanical cues and normal tissue homeostasis or diseases have been identified, the regulatory mechanisms among different mechanical cues are not yet comprehensively illustrated, and no effective therapies are currently available targeting mechanical cue-related signaling. This review systematically summarizes the characteristics and regulatory mechanisms of typical mechanical cues in normal conditions and diseases with the updated evidence. The key effectors responding to mechanical stimulations are listed, such as Piezo channels, integrins, Yes-associated protein (YAP) /transcriptional coactivator with PDZ-binding motif (TAZ), and transient receptor potential vanilloid 4 (TRPV4). We also reviewed the key signaling pathways, therapeutic targets and cutting-edge clinical applications of diseases related to mechanical cues.

The ER calcium channel Csg2 integrates sphingolipid metabolism with autophagy.

Sphingolipids are ubiquitous components of membranes and function as bioactive lipid signaling molecules. Here, through genetic screening and lipidomics analyses, we find that the endoplasmic reticulum (ER) calcium channel Csg2 integrates sphingolipid metabolism with autophagy by regulating ER calcium homeostasis in the yeast Saccharomyces cerevisiae. Csg2 functions as a calcium release channel and maintains calcium homeostasis in the ER, which enables normal functioning of the essential sphingolipid synthase Aur1. Under starvation conditions, deletion of Csg2 causes increases in calcium levels in the ER and then disturbs Aur1 stability, leading to accumulation of the bioactive sphingolipid phytosphingosine, which specifically and completely blocks autophagy and induces loss of starvation resistance in cells. Our findings indicate that calcium homeostasis in the ER mediated by the channel Csg2 translates sphingolipid metabolism into autophagy regulation, further supporting the role of the ER as a signaling hub for calcium homeostasis, sphingolipid metabolism and autophagy.

Phenotypic Heterogeneity Analysis of APC-Mutant Colon Cancer by Proteomics and Phosphoproteomics Identifies RAI14 as a Key Prognostic Determinant in East Asians and Westerners.

Adenomatous polyposis coli (APC) is an important tumor suppressor and is mostly linked to the regulation of the Wnt/ß-catenin signaling pathway. APC mutation has been identified as an early event in more than 80% of sporadic colorectal cancers (CRCs). Moreover, prognostic differences are observed in CRC patients with APC mutations. Although previous genomics studies have investigated the roles of concomitant gene mutations in determining the phenotypic heterogeneity of APC-mutant tumors, valuable prognostic determinants for APC-mutant CRC patients are still lacking. Based on the proteome and phosphoproteome data, we classified APC-mutant colon cancer patients and revealed genomic, proteomic, and phosphoproteomic heterogeneity in APC-mutant tumors. More importantly, we identified RAI14 as a key prognostic determinant for APC-mutant but not APC-wildtype colon cancer patients. The heterogeneity and the significance of prognostic biomarkers in APC-mutant tumors were further validated in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) colon cancer cohort. In addition, we found that colon cancer patients with high expression of RAI14 were less responsive to chemotherapy. Knockdown of RAI14 in cell lines led to reduced cell migration and changes in epithelial-mesenchymal transition (EMT)-related markers. Mechanistically, knockdown of RAI14 remodeled the phosphoproteome associated with cell adhesion, which might affect EMT marker expression and promote F-actin degradation. Collectively, this work describes the phenotypic heterogeneity of APC-mutant tumors and identifies RAI14 as an important prognostic determinant for APC-mutant colon cancer patients. The prognostic utility of RAI14 in APC-mutant colon cancer will provide early warning and increase the chance of successful treatment.

Targeting RNA G-quadruplex with repurposed drugs blocks SARS-CoV-2 entry.

The rapid emergence of SARS-CoV-2 variants of concern, the complexity of infection, and the functional redundancy of host factors, underscore an urgent need for broad-spectrum antivirals against the continuous COVID-19 pandemic, with drug repurposing as a viable therapeutic strategy. Here we report the potential of RNA G-quadruplex (RG4)-targeting therapeutic strategy for SARS-CoV-2 entry. Combining bioinformatics, biochemical and biophysical approaches, we characterize the existence of RG4s in several SARS-CoV-2 host factors. In silico screening followed by experimental validation identify Topotecan (TPT) and Berbamine (BBM), two clinical approved drugs, as RG4-stabilizing agents with repurposing potential for COVID-19. Both TPT and BBM can reduce the protein level of RG4-containing host factors, including ACE2, AXL, FURIN, and TMPRSS2. Intriguingly, TPT and BBM block SARS-CoV-2 pseudovirus entry into target cells in vitro and murine tissues in vivo. These findings emphasize the significance of RG4 in SARS-CoV-2 pathogenesis and provide a potential broad-spectrum antiviral strategy for COVID-19 prevention and treatment.

A vaccine based on the yeast-expressed receptor-binding domain (RBD) elicits broad immune responses against SARS-CoV-2 variants.

Development of safe and efficient vaccines is still necessary to deal with the COVID-19 pandemic. Herein, we reported that yeast-expressed recombinant RBD proteins either from wild-type or Delta SARS-CoV-2 were able to elicit immune responses against SARS-CoV-2 and its variants. The wild-type RBD (wtRBD) protein was overexpressed in Pichia pastoris, and the purified protein was used as the antigen to immunize mice after formulating an aluminium hydroxide (Alum) adjuvant. Three immunization programs with different intervals were compared. It was found that the immunization with an interval of 28 days exhibited the strongest immune response to SARS-CoV-2 than the one with an interval of 14 or 42 days based on binding antibody and the neutralizing antibody (NAb) analyses. The antisera from the mice immunized with wtRBD were able to neutralize the Beta variant with a similar efficiency but the Delta variant with 2~2.5-fold decreased efficiency. However, more NAbs to the Delta variant were produced when the Delta RBD protein was used to immunize mice. Interestingly, the NAbs may cross react with the Omicron variant. To increase the production of NAbs, the adjuvant combination of Alum and CpG oligonucleotides was used. Compared with the Alum adjuvant alone, the NAbs elicited by the combined adjuvants exhibited an approximate 10-fold increase for the Delta and a more than 53-fold increase for the Omicron variant. This study suggested that yeast-derived Delta RBD is a scalable and an effective vaccine candidate for SARS-CoV-2 and its variants.

Scramblases and virus infection.

The asymmetric distribution of lipids, maintained by flippases/floppases and scramblases, plays a pivotal role in various physiologic processes. Scramblases are proteins that move phospholipids between the leaflets of the lipid bilayer of the cellular membrane in an energy-independent manner. Recent studies have indicated that viral infection is closely related to cellular lipid distribution. The level and distribution of phosphatidylserine (PtdSer) in cells have been demonstrated to be critical regulators of viral infections. Previous studies have supported that the infection of human immunodeficiency virus (HIV), Zika virus, Ebola virus (EBOV), influenza virus, and dengue fever virus require the externalization of phospholipids mediated by scramblases, which are also involved in the pathogenicity of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we review the relationship of scramblases with viruses and the potential viral effector proteins that might utilize host scramblases.

Crosstalk of Synapsin1 palmitoylation and phosphorylation controls the dynamicity of synaptic vesicles in neurons.

The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.

Designing Overlap Extension PCR Primers for Protein Mutagenesis: A Programmatic Approach.

Overlap extension PCR is one of the routinely used methods to generate mutagenic genes for the functional and structural study of proteins. However, it is time-consuming to design the overlapping mutagenic primers and gene primers by manual operation. In this chapter, we present a Python script that is able to search all the possible primer combinations according to the preset definitions and calculate the necessary parameters of each primer for the users, which could facilitate the primer design process. Up to 256 pairs of primers can be provided for selection using this script.

Glycyrrhetinic acid restricts mitochondrial energy metabolism by targeting SHMT2.

Glycyrrhetinic acid (GA) is a natural product of licorice with mitochondria targeting properties and shows broad anticancer activities, but its targets and underlying mechanisms remain elusive. Here, we identified the mitochondrial enzyme serine hydroxymethyltransferase 2 (SHMT2) as a target of GA by using chemical proteomics. Binding to and inhibiting the activity of SHMT2 by GA were validated in vitro and in vivo. Knockout of SHMT2 or inhibiting SHMT2 with GA restricts mitochondrial energy supplies by downregulating mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid ß-oxidation, and consequently suppresses cancer cell proliferation and tumor growth. Crystal structures of GA derivatives indicate that GA occupies SHMT2 folate-binding pocket and regulates SHMT2 activity. Modifications at GA carboxylic group with diamines significantly improved its anticancer potency, demonstrating GA as a decent structural template for SHMT2 inhibitor development.

Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis.

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB-MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes.