TMPRSS2 and glycan receptors synergistically facilitate coronavirus entry.

The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.

A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

A method for predicting drugs that can boost the efficacy of immune checkpoint blockade.

Combination therapy is a promising therapeutic strategy to enhance the efficacy of immune checkpoint blockade (ICB); however, predicting drugs for effective combination is challenging. Here we developed a general data-driven method called CM-Drug for screening compounds that can boost ICB treatment efficacy based on core and minor gene sets identified between responsive and nonresponsive samples in ICB therapy. The CM-Drug method was validated using melanoma and lung cancer mouse models, with combined therapeutic efficacy demonstrated in eight of nine predicted compounds. Among these compounds, taltirelin had the strongest synergistic effect. Mechanistic analysis and experimental verification demonstrated that taltirelin can stimulate CD8+ T cells and is mediated by the induction of thyroid-stimulating hormone. This study provides an effective and general method for predicting and evaluating drugs for combination therapy and identifies candidate compounds for future ICB combination therapy.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Structure of the human ATP synthase.

Biological energy currency ATP is produced by F1Fo-ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the ß subunit of F1Fo-ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F1 and Fo motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex.

Inhibition of M. tuberculosis and human ATP synthase by BDQ and TBAJ-587.

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.

Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity.

CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Structure of the human respiratory complex II.

Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86 Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss.

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss.

Lowering systolic blood pressure to less than 120 mm Hg versus less than 140 mm Hg in patients with high cardiovascular risk with and without diabetes or previous stroke: an open-label, blinded-outcome, randomised trial.

BACKGROUND: Uncertainty exists about whether lowering systolic blood pressure to less than 120 mm Hg is superior to that of less than 140 mm Hg, particularly in patients with diabetes and patients with previous stroke. METHODS: In this open-label, blinded-outcome, randomised controlled trial, participants with high cardiovascular risk were enrolled from 116 hospitals or communities in China. We used minimised randomisation to assign participants to intensive treatment targeting standard office systolic blood pressure of less than 120 mm Hg or standard treatment targeting less than 140 mm Hg. The primary outcome was a composite of myocardial infarction, revascularisation, hospitalisation for heart failure, stroke, or death from cardiovascular causes, assessed by the intention-to-treat principle. This trial was registered with ClinicalTrials.gov, NCT04030234. FINDINGS: Between Sept 17, 2019, and July 13, 2020, 11 255 participants (4359 with diabetes and 3022 with previous stroke) were assigned to intensive treatment (n=5624) or standard treatment (n=5631). Their mean age was 64·6 years (SD 7·1). The mean systolic blood pressure throughout the follow-up (except the first 3 months of titration) was 119·1 mm Hg (SD 11·1) in the intensive treatment group and 134·8 mm Hg (10·5) in the standard treatment group. During a median of 3·4 years of follow-up, the primary outcome event occurred in 547 (9·7%) participants in the intensive treatment group and 623 (11·1%) in the standard treatment group (hazard ratio [HR] 0·88, 95% CI 0·78-0·99; p=0·028). There was no heterogeneity of effects by diabetes status, duration of diabetes, or history of stroke. Serious adverse events of syncope occurred more frequently in the intensive treatment group (24 [0·4%] of 5624) than in standard treatment group (eight [0·1%] of 5631; HR 3·00, 95% CI 1·35-6·68). There was no significant between-group difference in the serious adverse events of hypotension, electrolyte abnormality, injurious fall, or acute kidney injury. INTERPRETATION: For hypertensive patients at high cardiovascular risk, regardless of the status of diabetes or history of stroke, the treatment strategy of targeting systolic blood pressure of less than 120 mm Hg, as compared with that of less than 140 mm Hg, prevents major vascular events, with minor excess risk. FUNDING: The Ministry of Science and Technology of China and Fuwai Hospital. TRANSLATION: For the Mandarin translation of the abstract see Supplementary Materials section.

ICBcomb: a comprehensive expression database for immune checkpoint blockade combination therapy.

The success of immune checkpoint blockade (ICB) promotes the immunotherapy to be a new pillar in cancer treatment. However, the low response rate of the ICB therapy limits its application. To increase the response rate and enhance efficacy, the ICB combination therapy has emerged and its clinical trials are increasing. Nevertheless, the gene expression profile and its pattern of ICB combination were not comprehensively studied, which limits the understanding of the ICB combination therapy and the identification of new drugs. Here, we constructed ICBcomb (http://bioinfo.life.hust.edu.cn/ICBcomb/), a comprehensive database, by analyzing the human and mouse expression data of the ICB combination therapy and comparing them between groups treated with ICB, other drugs or their combinations. ICBcomb contains 1399 samples across 29 cancer types involving 52 drugs. It provides a user-friendly web interface for demonstrating the results of the available comparisons in the ICB combination therapy datasets with five functional modules: [1, 2] the 'Dataset/Disease' modules for browsing the expression, enrichment and comparison results in each dataset or disease; [3] the 'Gene' module for inputting a gene symbol and displaying its expression and comparison results across datasets/diseases; [4] the 'Gene Set' module for GSVA/GSEA enrichment analysis on the built-in gene sets and the user-input gene sets in different comparisons; [5] the 'Immune Cell' module for immune cell infiltration comparison between different groups by immune cell abundance analysis. The ICBcomb database provides the first resource for gene expression profile and comparison in ICB combination therapy, which may provide clues for discovering the mechanism of effective combination strategies and new combinatory drugs.

NAc-DBS corrects depression-like behaviors in CUMS mouse model via disinhibition of DA neurons in the VTA.

Major depressive disorder (MDD) is characterized by diverse debilitating symptoms that include loss of motivation and anhedonia. If multiple medications, psychotherapy, and electroconvulsive therapy fail in some patients with MDD, their condition is then termed treatment-resistant depression (TRD). MDD can be associated with abnormalities in the reward-system-dopaminergic mesolimbic pathway, in which the nucleus accumbens (NAc) and ventral tegmental area (VTA) play major roles. Deep brain stimulation (DBS) applied to the NAc alleviates the depressive symptoms of MDD. However, the mechanism underlying the effects of this DBS has remained elusive. In this study, using the chronic unpredictable mild stress (CUMS) mouse model, we investigated the behavioral and neurobiological effects of NAc-DBS on the multidimensional depression-like phenotypes induced by CUMS by integrating behavioral, in vivo microdialysis coupled with high-performance liquid chromatography-electrochemical detector (HPLC-ECD), calcium imaging, pharmacological, and genetic manipulation methods in freely moving mice. We found that long-term and repeated, but not single, NAc-DBS induced robust antidepressant responses in CUMS mice. Moreover, even a single trial NAc-DBS led to the elevation of the γ-aminobutyric acid (GABA) neurotransmitter, accompanied by the increase in dopamine (DA) neuron activity in the VTA. Both the inhibition of the GABAA receptor activity and knockdown of the GABAA-α1 gene in VTA-GABA neurons blocked the antidepressant effect of NAc-DBS in CUMS mice. Our results showed that NAc-DBS could disinhibit VTA-DA neurons by regulating the level of GABA and the activity of VTA-GABA in the VTA and could finally correct the depression-like behaviors in the CUMS mouse model.

Phase Change Thermal Storage Materials for Interdisciplinary Applications.

Functional phase change materials (PCMs) capable of reversibly storing and releasing tremendous thermal energy during the isothermal phase change process have recently received tremendous attention in interdisciplinary applications. The smart integration of PCMs with functional supporting materials enables multiple cutting-edge interdisciplinary applications, including optical, electrical, magnetic, acoustic, medical, mechanical, and catalytic disciplines etc. Herein, we systematically discuss thermal storage mechanism, thermal transfer mechanism, and energy conversion mechanism, and summarize the state-of-the-art advances in interdisciplinary applications of PCMs. In particular, the applications of PCMs in acoustic, mechanical, and catalytic disciplines are still in their infancy. Simultaneously, in-depth insights into the correlations between microscopic structures and thermophysical properties of composite PCMs are revealed. Finally, current challenges and future prospects are also highlighted according to the up-to-date interdisciplinary applications of PCMs. This review aims to arouse broad research interest in the interdisciplinary community and provide constructive references for exploring next generation advanced multifunctional PCMs for interdisciplinary applications, thereby facilitating their major breakthroughs in both fundamental researches and commercial applications.

METTL3 promotes trophoblast ferroptosis in preeclampsia by stabilizing the ACSL4 m6A modification.

This study aims to explore the role of methyltransferase-like 3 (METTL3) modulation of ferroptosis in the pathogenesis of trophoblast-mediated preeclampsia. The expression of METTL3 and acyl-CoA synthetase long chain family member 4 (ACSL4) was measured in clinical placental tissues and trophoblasts using qPCR and Western blot techniques. The effects of METTL3 on the symptoms of preeclampsia were also validated in rat models. METTL3 and ACSL4 were upregulated in placental tissues from patients with preeclampsia and in hypoxia-induced trophoblasts. METTL3 silencing increased the migration and invasion of trophoblasts cultured under hypoxic conditions. Knockdown of METTL3 increased cell viability and suppressed ferroptosis in hypoxia-stimulated trophoblasts. Hypoxia increased the level of m6A in cells, whereas silencing METTL3 partially reversed this change. Silencing METTL3 resulted in a decrease in m6A modification of ACSL4 mRNA, which led to a reduction in ACSL4 mRNA stability. ACSL4 upregulation partially reversed the effects of METTL3 silencing on cell viability, migration, invasion, and ferroptosis in hypoxia-stimulated trophoblasts. Inhibition of METTL3 in preeclampsia rats decreased blood pressure, urine protein levels, fetal survival rate, and ACSL4-mediated ferroptosis. METTL3 elevates ferroptosis to inhibit the migration and invasion of trophoblasts and in vivo preeclampsia symptoms by catalyzing the m6A modification of ACSL4 mRNA.

Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

METTL3 as a novel diagnosis and treatment biomarker and its association with glycolysis, cuproptosis and ceRNA in oesophageal carcinoma.

METTL3 has been shown to be involved in regulating a variety of biological processes. However, the relationship between METTL3 expression and glycolysis, cuproptosis-related genes and the ceRNA network in oesophageal carcinoma (ESCA) remains unclear. ESCA expression profiles from databases were obtained, and target genes were identified using differential analysis and visualization. Immunohistochemistry (IHC) staining assessed METTL3 expression differences. Functional enrichment analysis using GO, KEGG and GSEA was conducted on the co-expression profile of METTL3. Cell experiments were performed to assess the effect of METTL3 interference on tumour cells. Correlation and differential analyses were carried out to assess the relationship between METTL3 with glycolysis and cuproptosis. qRT-PCR was used to validate the effects of METTL3 interference on glycolysis-related genes. Online tools were utilized to screen and construct ceRNA networks based on the ceRNA theory. METTL3 expression was significantly higher in ESCA compared to the controls. The IHC results were consistent with the above results. Enrichment analysis revealed that METTL3 is involved in multiple pathways associated with tumour development. Significant correlations were observed between METTL3 and glycolysis-related genes and cuproptosis-related gene. Experiments confirmed that interfered with METTL3 significantly inhibited glucose uptake and lactate production in tumour cells, and affected the expression of glycolytic-related genes. Finally, two potential ceRNA networks were successfully predicted and constructed. Our study establishes the association between METTL3 overexpression and ESCA progression. Additionally, we propose potential links between METTL3 and glycolysis, cuproptosis and ceRNA, presenting a novel targeted therapy strategy for ESCA.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the ß-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than ß-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and ß2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo.