The role of deubiquitinases in cardiac disease.

Deubiquitinases are a group of proteins that identify and digest monoubiquitin chains or polyubiquitin chains attached to substrate proteins, preventing the substrate protein from being degraded by the ubiquitin-proteasome system. Deubiquitinases regulate cellular autophagy, metabolism and oxidative stress by acting on different substrate proteins. Recent studies have revealed that deubiquitinases act as a critical regulator in various cardiac diseases, and control the onset and progression of cardiac disease through a board range of mechanism. This review summarizes the function of different deubiquitinases in cardiac disease, including cardiac hypertrophy, myocardial infarction and diabetes mellitus-related cardiac disease. Besides, this review briefly recapitulates the role of deubiquitinases modulators in cardiac disease, providing the potential therapeutic targets in the future.

Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

Mechanism of millisecond Lys48-linked poly-ubiquitin chain formation by cullin-RING ligases.

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

Breaking the K48-chain: linking ubiquitin beyond protein degradation.

The discovery of ubiquitin conjugation to lysines and the role of K48-linked polyubiquitin in targeting substrates for proteasomal degradation was followed by revelation of non-degradative roles of ubiquitination and, more recently, of non-canonical covalent ubiquitin linkages. Here we summarize findings of the ever-expanding array of ubiquitin signals and their biological roles.

Simultaneous substrate and ubiquitin modification recognition by bispecific antibodies enables detection of ubiquitinated RIP1 and RIP2.

Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.

Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.

The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.

Bacterial ligases reveal fundamental principles of polyubiquitin specificity.

Homologous to E6AP C terminus (HECT) E3 ubiquitin (Ub) ligases direct substrates toward distinct cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal attached. How polyUb specificity is achieved has been a long-standing mystery, despite extensive study in various hosts, ranging from yeast to human. The bacterial pathogens enterohemorrhagic Escherichia coli and Salmonella Typhimurium encode outlying examples of "HECT-like" (bHECT) E3 ligases, but commonalities to eukaryotic HECT (eHECT) mechanism and specificity had not been explored. We expanded the bHECT family with examples in human and plant pathogens. Three bHECT structures in primed, Ub-loaded states resolved key details of the entire Ub ligation process. One structure provided a rare glimpse into the act of ligating polyUb, yielding a means to rewire polyUb specificity of both bHECT and eHECT ligases. Studying this evolutionarily distinct bHECT family has revealed insight into the function of key bacterial virulence factors as well as fundamental principles underlying HECT-type Ub ligation.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in rats.

BACKGROUND: Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. METHODS: We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. RESULTS: We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. CONCLUSIONS: These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).

Male and female brains have differences in size, development, and cellular processes. Further, males and females have differences in likelihood of developing certain anxiety-related disorders, such as post-traumatic stress disorder (PTSD). We previously observed sex differences in a cellular mechanism that controls the destruction of proteins via tagging by the protein modifier ubiquitin in resting and behaviorally trained animals. We found that adult female rats "ubiquitinated" different proteins during learning and had more ubiquitin than male rats at rest in the amygdala, the brain region that controls emotional regulation. This study investigated if the sex difference in ubiquitin at rest changed as animals age, including the proteins being ubiquitinated and how the amount of ubiquitin was controlled. We also investigated if male and female rats need ubiquitin for memory formation. We found that males and females ubiquitinate different proteins, but that aging also contributes to changes in this, suggesting that sexual maturity may be important for controlling the amount of ubiquitin in females. Lastly, we found that only female rats needed ubiquitin in the amygdala for forming a fear memory. These results are important for understanding the role of ubiquitin activity at different developmental stages and for forming fear-based memories in both sexes. Since females are more likely to develop PTSD than males, these data could help understand how different cellular processes work together in PTSD development to create better treatment options.

ß-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to ßarrestins. In addition, ßarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an in vitro reconstituted system and show that ßarrestin1 (ßarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that ßarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the ßarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.

DLG1 promotes the antiviral innate immune response by inhibiting p62-mediated autophagic degradation of IKKε.

IMPORTANCE: The type-I interferon (IFN-I) signaling pathway is the first line of antiviral innate immunity. It must be precisely regulated against virus-induced damage. The tightly regulated mechanisms of action of host genes in the antiviral innate immune signaling pathway are still worth studying. Here, we report a novel role of DLG1 in positively regulating the IκB kinase epsilon (IKKε)-mediated IFN-I signaling response against negative-stranded RNA virus replication, whereas the RNA virus inhibits the expression of DLG1 for immune escape. Importantly, the E3 ligase March2 interacts with and promotes K27-linked polyubiquitination of IKKε, and p62 is a cargo receptor that recognizes ubiquitinated IKKε for eventual autophagic degradation. Together, the current findings elucidate the role of DLG1 in the antiviral IFN-I signaling pathway and viral infection repression.

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units.

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

Epitope identification of a Lys63 linkage ubiquitin antibody by mass spectrometric epitope excision and extraction approaches.

Ubiquitin, a conserved protein in eukaryotic cells, exists as a monomer or polyubiquitin chains known as isopeptide-linked polymers. These chains are attached to a substrate or other ubiquitin molecules through a covalent bond between the α-amino group of lysine in ubiquitin and glycine in the C-terminal of the subsequent ubiquitin unit. The choice of the specific lysine residue in ubiquitin for forming ubiquitin-ubiquitin chains determines its biochemical and biological function. A detailed chemical structure-function evaluation of the respective polyubiquitin chain is required. Interestingly, specific lysine linkage polyubiquitin chains become covalently bonded to many pathological inclusions seen in serious human disease states which appear to be resistant to normal degradation, so the interaction between polyubiquitin chains and ubiquitin antibodies is very useful. For example, the neurofibrillary tangles of Alzheimer's disease and the Lewy bodies seen in Parkinson's disease are heavily ubiquitinated and can be readily visualized using specific ubiquitin antibodies. This study utilized synthetic ubiquitin building block peptides that contained various lysine residues (K6, K11, K33, K48, and K63) linked to a Gly-Gly dipeptide, with the aim of exploring the recognition specificity of the Lys63-polyubiquitin antibody. The interaction studies between different ubiquitin building blocks and the specific Lys63-ubiquitin (K63-Ub) antibody were performed by affinity-mass spectrometry (Affinity-MS) and immunoblotting which enables direct protein identification from biological material with unprecedented selectivity. Affinity-MS and dot blot data proved the specific binding of the K63-Ub antibody to the ubiquitin peptides containing Lys6 or Lys63 residues. In epitope excision for mass spectrometric epitope identification, the ubiquitin building block with Lys63 residue bound to the immobilized K63-Ub antibody was proteolytically cleaved using pronase. The resulting epitope and non-epitope fractions were subjected to matrix-assisted laser desorption/ionization-time of flight analysis, revealing that the epitope is located within the sequence ubiquitin(60-66). Epitope extraction-MS consistently confirmed these findings.

Myddosome clustering in IL-1 receptor signaling regulates the formation of an NF-kB activating signalosome.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade.

The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor (RLR) signaling cascade. Lys63 (K63)-linked polyubiquitination is critical for MAVS aggregation, yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes (DUBs) remain elusive. Here, we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with the prerequisite of the CARDRIG-I-CARDMAVS interaction. Interestingly, many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage. We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS. In addition, we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS. Consistently, USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon. Mice with a deficiency in USP10 show more potent resistance to RNA virus infection. Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.

The pros and cons of ubiquitination on the formation of protein condensates.

Ubiquitination, a post-translational modification that attaches one or more ubiquitin (Ub) molecules to another protein, plays a crucial role in the phase-separation processes. Ubiquitination can modulate the formation of membrane-less organelles in two ways. First, a scaffold protein drives phase separation, and Ub is recruited to the condensates. Second, Ub actively phase-separates through the interactions with other proteins. Thus, the role of ubiquitination and the resulting polyUb chains ranges from bystanders to active participants in phase separation. Moreover, long polyUb chains may be the primary driving force for phase separation. We further discuss that the different roles can be determined by the lengths and linkages of polyUb chains which provide preorganized and multivalent binding platforms for other client proteins. Together, ubiquitination adds a new layer of regulation for the flow of material and information upon cellular compartmentalization of proteins.

PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis.

Ubiquitination modulates protein turnover or activity depending on the number and location of attached ubiquitin (Ub) moieties. Proteins marked by a lysine 48 (K48)-linked polyubiquitin chain are usually targeted to the 26S proteasome for degradation; however, other polyubiquitin chains, such as those attached to K63, usually regulate other protein properties. Here, we show that 2 PLANT U-BOX E3 ligases, PUB25 and PUB26, facilitate both K48- and K63-linked ubiquitination of the transcriptional regulator INDUCER OF C-REPEAT BINDING FACTOR (CBF) EXPRESSION1 (ICE1) during different periods of cold stress in Arabidopsis (Arabidopsis thaliana), thus dynamically modulating ICE1 stability. Moreover, PUB25 and PUB26 attach both K48- and K63-linked Ub chains to MYB15 in response to cold stress. However, the ubiquitination patterns of ICE1 and MYB15 mediated by PUB25 and PUB26 differ, thus modulating their protein stability and abundance during different stages of cold stress. Furthermore, ICE1 interacts with and inhibits the DNA-binding activity of MYB15, resulting in an upregulation of CBF expression. This study unravels a mechanism by which PUB25 and PUB26 add different polyubiquitin chains to ICE1 and MYB15 to modulate their stability, thereby regulating the timing and degree of cold stress responses in plants.

LUBAC-mediated linear ubiquitination in tissue homeostasis and disease.

In addition to its role in the ubiquitin-proteasome system of protein degradation, polyubiquitination is involved in the regulation of intracellular events. Depending on the type of ubiquitin-ubiquitin linkage used, polyubiquitin can assume several types of structures. The spatiotemporal dynamics of polyubiquitin involve multiple adaptor proteins and induce different downstream outputs. Linear ubiquitination, in which the N-terminal methionine on the acceptor ubiquitin serves as the site for ubiquitin-ubiquitin conjugation, is a rare and atypical type of polyubiquitin modification. The production of linear ubiquitin chains is dependent on various external inflammatory stimuli and leads to the transient activation of the downstream NF-κB signalling pathway. This in turn suppresses extrinsic programmed cell death signals and protects cells from activation-induced cell death under inflammatory conditions. Recent evidence has revealed the role of linear ubiquitination in various biological processes under both physiological and pathological conditions. This led us to propose that linear ubiquitination may be pivotal in the 'inflammatory adaptation' of cells, and consequently in tissue homeostasis and inflammatory disease. In this review, we focused on the physiological and pathophysiological roles of linear ubiquitination in vivo in response to a changing inflammatory microenvironment.

The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.