scapGNN: A graph neural network-based framework for active pathway and gene module inference from single-cell multi-omics data.

Although advances in single-cell technologies have enabled the characterization of multiple omics profiles in individual cells, extracting functional and mechanistic insights from such information remains a major challenge. Here, we present scapGNN, a graph neural network (GNN)-based framework that creatively transforms sparse single-cell profile data into the stable gene-cell association network for inferring single-cell pathway activity scores and identifying cell phenotype-associated gene modules from single-cell multi-omics data. Systematic benchmarking demonstrated that scapGNN was more accurate, robust, and scalable than state-of-the-art methods in various downstream single-cell analyses such as cell denoising, batch effect removal, cell clustering, cell trajectory inference, and pathway or gene module identification. scapGNN was developed as a systematic R package that can be flexibly extended and enhanced for existing analysis processes. It provides a new analytical platform for studying single cells at the pathway and network levels.

Quantitative phosphoproteomic profiling of mouse sperm maturation in epididymis revealed kinases important for sperm motility.

Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6,447 phosphorylation sites in 1,407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes.

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 µg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.

DeepSCP: utilizing deep learning to boost single-cell proteome coverage.

Multiplexed single-cell proteomes (SCPs) quantification by mass spectrometry greatly improves the SCP coverage. However, it still suffers from a low number of protein identifications and there is much room to boost proteins identification by computational methods. In this study, we present a novel framework DeepSCP, utilizing deep learning to boost SCP coverage. DeepSCP constructs a series of features of peptide-spectrum matches (PSMs) by predicting the retention time based on the multiple SCP sample sets and fragment ion intensities based on deep learning, and predicts PSM labels with an optimized-ensemble learning model. Evaluation of DeepSCP on public and in-house SCP datasets showed superior performances compared with other state-of-the-art methods. DeepSCP identified more confident peptides and proteins by controlling q-value at 0.01 using target-decoy competition method. As a convenient and low-cost computing framework, DeepSCP will help boost single-cell proteome identification and facilitate the future development and application of single-cell proteomics.

Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility.

BACKGROUND: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized. RESULTS: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa-/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs. CONCLUSIONS: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

DeepSP: A Deep Learning Framework for Spatial Proteomics.

The study of protein subcellular localization (PSL) is a fundamental step toward understanding the mechanism of protein function. The recent development of mass spectrometry (MS)-based spatial proteomics to quantify the distribution of proteins across subcellular fractions provides us a high-throughput approach to predict unknown PSLs based on known PSLs. However, the accuracy of PSL annotations in spatial proteomics is limited by the performance of existing PSL predictors based on traditional machine learning algorithms. In this study, we present a novel deep learning framework named DeepSP for PSL prediction of an MS-based spatial proteomics data set. DeepSP constructs the new feature map of a difference matrix by capturing detailed changes between different subcellular fractions of protein occupancy profiles and uses the convolutional block attention module to improve the prediction performance of PSL. DeepSP achieved significant improvement in accuracy and robustness for PSL prediction in independent test sets and unknown PSL prediction compared to current state-of-the-art machine learning predictors. As an efficient and robust framework for PSL prediction, DeepSP is expected to facilitate spatial proteomics studies and contributes to the elucidation of protein functions and the regulation of biological processes.

Homeodomain-interacting protein kinase HIPK4 regulates phosphorylation of manchette protein RIMBP3 during spermiogenesis.

Nonobstructive azoospermia (NOA) is the most serious form of spermatogenesis abnormalities in male infertility. Genetic factors are important to consider as elements leading to NOA. Although many pathogenic genes have been reported, the causative genes of NOA for many patients are still unknown. In this study, we found ten point mutations in the gene encoding homeodomain-interacting protein kinase 4 (HIPK4) in patients with NOA, and using in vitro studies, we determined a premature termination point mutation (p. Lys490∗, c.1468A>T) that can cause decreased expression of HIPK4. Our phosphoproteomic analysis of Hipk4-/- testes revealed phosphorylation of multiple proteins regulated by HIPK4 during spermiogenesis. We also confirmed that a substrate of HIPK4 with four downregulated phosphorylation sites matching the xSPx motif is the known manchette-related protein RIMS-binding protein 3, which is required for sperm head morphogenesis. Therefore, we conclude HIPK4 regulates the phosphorylation of manchette protein RIMS-binding protein 3 and plays essential roles in sperm head shaping and male fertility.

Global phosphoproteomic analysis identified key kinases regulating male meiosis in mouse.

Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells.

Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon injury, MuSCs exit quiescence in vivo to become activated, re-enter the cell cycle to proliferate, and differentiate to repair the damaged muscles. It remains unclear which extrinsic cues and intrinsic signaling pathways regulate quiescence exit during MuSC activation. Here, we demonstrated that inducible MuSC-specific deletion of p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), rendered MuSCs unable to exit quiescence, resulting in severely impaired MuSC proliferation and muscle regeneration. Genetic reactivation of mTORC1, or knockdown of FoxOs, in p110α-null MuSCs partially rescued the above defects, making them key effectors downstream of PI3K in regulating quiescence exit. c-Jun was found to be a key transcriptional target of the PI3K/mTORC1 signaling axis essential for MuSC quiescence exit. Moreover, induction of a constitutively active PI3K in quiescent MuSCs resulted in spontaneous MuSC activation in uninjured muscles and subsequent depletion of the MuSC pool. Thus, PI3K-p110α is both necessary and sufficient for MuSCs to exit quiescence in response to activating signals.

Recent progress of proteomic analysis on spermatogenesis†.

Testis, the only organ responsible for generating sperm, is by far the organ with the largest variety of proteins and tissue-specific proteins in humans. In testis, spermatogenesis is a multi-step complex process well-accepted that protein and mRNA are decoupled in certain stages of spermatogenesis. With the fast development of mass spectrometry-based proteomics, it is possible to systemically study protein abundances and modifications in testis and sperm to help us understand the molecular mechanisms of spermatogenesis. This review provides an overview of the recent progress of proteomics analysis on spermatogenesis, including protein expression and multiple post-translational modifications, such as phosphorylation, glycosylation, ubiquitylation, and acetylation.

Systematic analysis of the ubiquitome in mouse testis.

A growing body of evidence now supports the fact that protein ubiquitination is an important modification during the regulation of spermatogenesis. However, little is known about the ubiquitome of the testis. In this study, we created a large-scale mouse testis ubiquitome profile using di-glycine remnant antibodies and mass spectrometry and identified a total of 14,219 ubiquitination sites in 4217 proteins. Bioinformatics and phenotypic analyses showed that the ubiquitinated proteins were closely related to meiosis and spermiogenesis. And 512 ubiquitination regulatory enzymes were identified in testis that can exert regulatory functions over ubiquitination: the homologous to E6AP C-terminus (HECT) and multi-subunit RING-finger type E3 ligases were significantly enriched. In addition, we identified 22 new ubiquitination sites on testicular histones and 146 ubiquitinated epigenetic factors, thus demonstrating that ubiquitination plays an important role in epigenetic regulation. Collectively, this in-depth characterization of the ubiquitome in mouse testis could provide a rich resource for further studies of regulatory events at the protein level during spermatogenesis. All MS data are available via ProteomeXchange with the identifier PXD025866.

Cyclin-dependent kinase 7 is essential for spermatogenesis by regulating retinoic acid signaling pathways and the STAT3 molecular pathway.

Spermatogenesis is a complex process that requires precise regulation. Phosphorylation plays a role in spermatogenesis by regulating protein structure and activity. This study focused on cyclin-dependent kinase 7 (CDK7), and explored its function and molecular mechanisms in spermatogenesis in vitro in a cell line and in vivo in a mouse model. Inhibition of CDK7 activity affected spermatogonia proliferation and differentiation, and we found that CDK7 regulates retinoic acid (RA)-mediated c-KIT expression to play a role in spermatogonia. Then, we demonstrated that inhibition of CDK7 affected meiosis initiation, DNA repair, and synaptonemal complex formation in meiosis progression, and CDK7 played this role by regulating RA-mediated STRA8 and REC8 signaling pathways. Moreover, inhibition of CDK7 impacted spermatid differentiation and resulted in decreased counts, decreased motility, and increased head deformity of sperm. We demonstrated that CDK7 affects germ cell apoptosis and sperm motility by activating STAT3 and that STAT3 further regulates Cortactin expression to influence the nuclear elongation, chromatin condensation, and acrosome formation of sperm. Additionally, EP300 was identified as another potential target phosphorylated by CDK7 that participates in chromatin condensation. Our results demonstrated the important role of CDK7 in all key aspects of spermatogenesis, potentially providing an effective target for clinical diagnosis and pathogenesis.

STYXL1 regulates CCT complex assembly and flagellar tubulin folding in sperm formation.

Tubulin-based microtubule is a core component of flagella axoneme and essential for sperm motility and male fertility. Structural components of the axoneme have been well explored. However, how tubulin folding is regulated in sperm flagella formation is still largely unknown. Here, we report a germ cell-specific co-factor of CCT complex, STYXL1. Deletion of Styxl1 results in male infertility and microtubule defects of sperm flagella. Proteomic analysis of Styxl1-/- sperm reveals abnormal downregulation of flagella-related proteins including tubulins. The N-terminal rhodanese-like domain of STYXL1 is important for its interactions with CCT complex subunits, CCT1, CCT6 and CCT7. Styxl1 deletion leads to defects in CCT complex assembly and tubulin polymerization. Collectively, our findings reveal the vital roles of germ cell-specific STYXL1 in CCT-facilitated tubulin folding and sperm flagella development.

Single-cell chromatin accessibility profiling reveals a self-renewing muscle satellite cell state.

A balance between self-renewal and differentiation is critical for the regenerative capacity of tissue-resident stem cells. In skeletal muscle, successful regeneration requires the orchestrated activation, proliferation, and differentiation of muscle satellite cells (MuSCs) that are normally quiescent. A subset of MuSCs undergoes self-renewal to replenish the stem cell pool, but the features that identify and define self-renewing MuSCs remain to be elucidated. Here, through single-cell chromatin accessibility analysis, we reveal the self-renewal versus differentiation trajectories of MuSCs over the course of regeneration in vivo. We identify Betaglycan as a unique marker of self-renewing MuSCs that can be purified and efficiently contributes to regeneration after transplantation. We also show that SMAD4 and downstream genes are genetically required for self-renewal in vivo by restricting differentiation. Our study unveils the identity and mechanisms of self-renewing MuSCs, while providing a key resource for comprehensive analysis of muscle regeneration.

Muscle satellite cells are impaired in type 2 diabetic mice by elevated extracellular adenosine.

Muscle regeneration is known to be defective under diabetic conditions. However, the underlying mechanisms remain less clear. Adult quiescent muscle satellite cells (MuSCs) from leptin-receptor-deficient (i.e., db/db) diabetic mice are defective in early activation in vivo, but not in culture, suggesting the involvement of pathogenic niche factors. Elevated extracellular adenosine (eAdo) and AMP (eAMP) are detected under diabetic conditions. eAdo and eAMP potently inhibit cell cycle re-entry of quiescent MuSCs and injury-induced muscle regeneration. Mechanistically, eAdo and eAMP engage the equilibrative Ado transporters (ENTs)-Ado kinase (ADK)-AMPK signaling axis in MuSCs to inhibit the mTORC1-dependent cell growth checkpoint. eAdo and eAMP also inhibit early activation of quiescent fibroadipogenic progenitors and human MuSCs by the same mechanism. Treatment of db/db diabetic mice with an ADK inhibitor partially rescues the activation defects of MuSCs in vivo. Thus, both ADK and ENTs represent potential therapeutic targets for restoring the regenerative functions of tissue stem cells in patients with diabetes.

Structure of BAI1/ELMO2 complex reveals an action mechanism of adhesion GPCRs via ELMO family scaffolds.

The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G protein-coupled receptors (aGPCRs) plays crucial roles in diverse cellular processes including phagocytosis, myoblast fusion, and synaptic development through the ELMO/DOCK/Rac signaling pathway, although the underlying molecular mechanism is not well understood. Here, we demonstrate that an evolutionarily conserved fragment located in the C-terminal cytoplasmic tail of BAI-aGPCRs is specifically recognized by the RBD-ARR-ELMO (RAE) supramodule of the ELMO family scaffolds. The crystal structures of ELMO2-RAE and its complex with BAI1 uncover the molecular basis of BAI/ELMO interactions. Based on the complex structure we identify aGPCR-GPR128 as another upstream receptor for the ELMO family scaffolds, most likely with a recognition mode similar to that of BAI/ELMO interactions. Finally, we map disease-causing mutations of BAI and ELMO and analyze their effects on complex formation.

Label-free multimodal nonlinear optical microscopy reveals fundamental insights of skeletal muscle development.

We developed a label-free nonlinear optical (NLO) microscope integrating the stimulated Raman scattering, multi-color two-photon excited fluorescence and second harmonic generation. The system produces multimodal images of protein content, mitochondria distribution and sarcomere structure of fresh muscle samples. With the advanced imaging technique, we studied the mal-development of skeletal muscle caused by sarcomeric gene deficiency. In addition, important development processes of normal muscle from neonatal to adult stage were also clearly revealed based on the changing sarcomere structure, mitochondria distribution and muscle fiber size. The results demonstrate that the newly developed multimodal NLO microscope is a powerful tool to assess the muscle integrity and function.