High Molecular Weight Complexes Research Articles

Enhancing spinnability and properties of carbon fibers through modification of isotropic coal tar pitch precursor

Producing general-purpose carbon fibers (GPCFs) with excellent properties using an economical raw material such as coal tar pitch (CTP) holds significant importance. To synthesize carbon fibers, the spinnability of precursor material plays an important role. However, the CTP impedes flowability during the spinning process due to possessing high molecular weight complex aromatic structures. To address this limitation, CTP was blended with varying proportions of petroleum pitch (5, 10 and 20 %) and spinnability and flow behavior were analyzed using Rheology measurements. The distinct constitutional properties of petroleum pitch also contributed to alterations in both the stabilization and carbonization processes. Carbon fibers produced from 10PP showed the highest tensile strength and modulus of ∼784 MPa and ∼47 GPa, respectively. The impact of oxidative and carbonization treatments on the resultant microstructural and mechanical properties of carbon fibers was thoroughly examined using various techniques such as FTIR, XPS, XRD, SEM, Raman and UTM. The results showed that the composition of petroleum pitch influenced the stabilization of fibers which eventually affected the carbonization process and resultant properties of carbon fibers. This study offers an understanding of the connection between the precursor's composition, stabilization conditions, and mechanical properties of the resultant carbon fibers.

Reduced protein kinase C delta in a high molecular weight complex in mitochondria and elevated creatine uptake into Barth syndrome B lymphoblasts

Reduced protein kinase C delta in a high molecular weight complex in mitochondria and elevated creatine uptake into Barth syndrome B lymphoblasts

Isotope-Edited Variable Temperature Infrared Spectroscopy for Measuring Transition Temperatures of Single A-T Watson-Crick Base Pairs in DNA Duplexes.

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this "sub-molecular scale thermometer", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.

Retraction Note: Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity

Retraction Note: Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Combining BNCT with carbonic anhydrase inhibition for mesothelioma treatment: Synthesis, in vitro, in vivo studies of ureidosulfamido carboranes

Mesothelioma is a malignant neoplasm of mesothelial cells caused by exposure to asbestos. The average survival time after diagnosis is usually nine/twelve months. A multi-therapeutic approach is therefore required to treat and prevent recurrence. Boronated derivatives containing a carborane cage, a sulfamido group and an ureido functionality (CA-USF) have been designed, synthesised and tested, in order to couple Boron Neutron Capture Therapy (BNCT) and the inhibition of Carbonic Anhydrases (CAs), which are overexpressed in many tumours. In vitro studies showed greater inhibition than the reference drug acetazolamide (AZ). To increase solubility in aqueous media, CA-USFs were used as inclusion complexes of hydroxypropyl β-cyclodextrin (HP-β-CD) in all the inhibition and cell experiments. BNCT experiments carried out on AB22 (murine mesothelioma) cell lines showed a marked inhibition of cell proliferation by CA-USFs, and in one case a complete inhibition of proliferation twenty days after neutron irradiation. Finally, in vivo neutron irradiation experiments on a mouse model of mesothelioma demonstrated the efficiency of combining CA IX inhibition and BNCT treatment. Indeed, a greater reduction in tumour mass was observed in treated mice compared to untreated mice, with a significant higher effect when combined with BNCT. For in vivo experiments CA-USFs were administered as inclusion complexes of higher molecular weight β-CD polymers thus increasing the selective extravasation into tumour tissue and reducing clearance. In this way, boron uptake was maximised and CA-USFs demonstrated to be in vivo well tolerated at a therapeutic dose. The therapeutic strategy herein described could be expanded to other cancers with increased CA IX activity, such as melanoma, glioma, and breast cancer.

Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

A candidate glycoconjugate vaccine induces protective antibodies in the serum and intestinal secretions, antibody recall response and memory T cells and protects against both typhoidal and non-typhoidal Salmonella serovars.

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium O-specific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and non-typhoidal Salmonella serovars.

Specialized digestive mechanism for an insect-bacterium gut symbiosis.

In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis.

RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

S-nitrosylation switches the Arabidopsis redox sensor protein, QSOX1, from an oxidoreductase to a molecular chaperone under heat stress

The Arabidopsis quiescin sulfhydryl oxidase 1 (QSOX1) thiol-based redox sensor has been identified as a negative regulator of plant immunity. Here, we have found that small molecular weight proteins of QSOX1 were converted to high molecular weight (HMW) complexes upon exposure to heat stress and that this was accompanied by a switch in QSOX1 function from a thiol-reductase to a molecular chaperone. Plant treatment with S-nitrosoglutathione (GSNO), which causes nitrosylation of cysteine residues (S-nitrosylation), but not with H2O2, induced HMW QSOX1 complexes. Thus, functional switching of QSOX1 is induced by GSNO treatment. Accordingly, simultaneous treatment of plants with heat shock and GSNO led to a significant increase in QSOX1 chaperone activity by increasing its oligomerization. Consequently, transgenic Arabidopsis overexpressing QSOX1 (QSOX1OE) showed strong resistance to heat shock, whereas qsox1 knockout plants exhibited high sensitivity to heat stress. Plant treatment with GSNO under heat stress conditions increased their resistance to heat shock. We conclude that S-nitrosylation allows the thiol-based redox sensor, QSOX1, to respond to various external stresses in multiple ways.

Functional changes of OsTrxm from reductase to molecular chaperone under heat shock stress

Ubiquitous disulfide reductases, thioredoxins (Trxs), function in the redox balance of all living organisms. Although the roles of the rice (Oryza sativa) Trx m-type isoform (OsTrxm) in chloroplast development have been already published, biochemical and molecular functions of OsTrxm remain to be elucidated for decades. The OsTrxm and its two conserved active cysteine mutant (OsTrxm C95S/C98S, referred to as OsTrxmC/S) proteins in Arabidopsis thaliana were overexpressed to characterize in vivo roles of active cysteines of OsTrxm. Interestingly, the OsTrxm overexpressed variant plants were resistant to heat shock treatment. Especially OsTrxmC/S with higher molecular weight (HMW) complexes showed higher heat tolerance than OsTrxm with lower molecular weight (LMW) structure in Arabidopsis thaliana. To confirm the importance of active cysteines on structural changes under heat stress, OsTrxm and OsTrxmC/S proteins were bacterially expressed and isolated. This study found that two proteins have various structures ranging from LMW to HMW complexes and have potential functions as a disulfide reductase and a molecular chaperone, which has never been reported anywhere. The function of molecular chaperone predominated in the HMW complexes, whereas the disulfide reductase function was observed in LMW forms. These results suggest that the active cysteines of OsTrxm play a critical role in protein structural change as well as heat tolerance in plants.

APP family member dimeric complexes are formed predominantly in synaptic compartments

BackgroundThe amyloid precursor protein (APP), a key player in Alzheimer’s disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions.ResultsWe investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls.ConclusionsTogether, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

NLRP11 is a pattern recognition receptor for bacterial lipopolysaccharide in the cytosol of human macrophages.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes.

Elucidation of the Binding Mechanism of Anionic Phospholipids to Antioxidant Protein Peroxiredoxin 2.

Peroxiredoxins (Prxs) belong to a family of ubiquitously expressed peroxidases that detoxify reactive oxygen species. In addition to their enzymatic function, Prxs also function as molecular chaperones. This functional switch is related to their degree of oligomerization. We have previously revealed that Prx2 interacts with anionic phospholipids and that the anionic phospholipid-containing Prx2 oligomer forms a high molecular weight (HMW) complex in a nucleotide-dependent manner. However, the detailed mechanism of the oligomer and HMW complex formation remains unclear. In this study, we investigated the anionic phospholipid binding site in Prx2 using site-directed mutagenesis to understand the mechanism of the oligomer formation. Our findings demonstrated that six binding site residues in Prx2 are important for the binding of anionic phospholipids.

IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition.

The Sec1–Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B

Loss-of-function variants of vacuolar protein sorting proteins VPS33B and VPS16B (VIPAS39) are causative for arthrogryposis, renal dysfunction, and cholestasis syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1-Munc18 protein family and thought to facilitate vesicular fusion via interaction with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, like its paralog VPS33A in the homotypic fusion and vacuole sorting complex. VPS33B and VPS16B are known to associate, but little is known about the composition, structure, or function of the VPS33B-VPS16B complex. We show here that human VPS33B-VPS16B is a high molecular weight complex, which we expressed in yeast to perform structural, composition, and stability analysis. Circular dichroism data indicate VPS33B-VPS16B has a well-folded α-helical secondary structure, and size-exclusion chromatography-multiangle light scattering revealed a molecular weight of ∼315kDa. Quantitative immunoblotting indicated a VPS33B:VPS16B ratio of 2:3. Expression of arthrogryposis, renaldysfunction, and cholestasis syndrome-causing VPS33B missense variants showed L30P disrupts complex formation but not S243F or H344D. Truncated VPS16B (amino acids 143 to 316) was sufficient to form a complex with VPS33B. Small-angle X-ray scattering and negative-staining EM revealed a two-lobed shape for VPS33B-VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. We propose a structure for VPS33B-VPS16B that allows the VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus associated membrane components. These observations reveal the only known potentially bidirectional Sec1-Munc18 protein complex.

Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. This study aimed to identify cysteine-rich secretory protein 2 interacting partners.These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction. The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2in pig sperm. Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2was present within a ∼150kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53kDa) and Aacrosin binding proteinunder all three conditions and additionally to acrosin (∼35kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.