Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Site-specific photo-crosslinking of Hsc70 with the KFERQ pentapeptide motif in a chaperone-mediated autophagy and microautophagy substrate in mammalian cells.

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.

Site-specific photo-crosslinking/cleavage for protein-protein interface identification reveals oligomeric assembly of lysosomal-associated membrane protein type 2A in mammalian cells.

Genetic code expansion enables site-specific photo-crosslinking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε -allyloxycarbonyl-α-hydroxyl-l-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal-associated membrane protein type 2A (LAMP2A), a receptor of chaperone-mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site-specific crosslinking and site-specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries.

A Quantitative Prediction Method for the Human Pharmacokinetics of Fc-Fusion Proteins.

BACKGROUND AND OBJECTIVE: Fc fusion is an effective strategy for extending the half-lives of therapeutic proteins. This study aimed to evaluate the applicability of a human pharmacokinetics prediction method for Fc-fusion proteins by extending on reported methods for monoclonal antibodies (mAbs). METHODS: To predict human pharmacokinetic profiles following intravenous (IV) dosing, the pharmacokinetic data for 11 Fc-fusion proteins in monkeys were analysed by two approaches: a species-invariant time method with a range of allometric exponents in clearance (CL, 0.7-1.0) and a two-compartment model reported for mAbs. The pharmacokinetic profiles following subcutaneous (SC) dosing were predicted by simple dose normalisation from monkeys or using the geometric means of the absorption rate constant (Ka) and bioavailability (BA) for mAbs or Fc-fusion proteins in humans and compared. RESULTS: In the case of IV administration, the area under the curve could be predicted for more than 85% of Fc-fusion proteins within a twofold difference from the observed value using the species-invariant time method (scaling exponent for CL, 0.95). For SC dosing, incorporating the geometric means of absorption parameters for both mAbs (BA 68.2%, Ka 0.287 day-1) and Fc-fusion proteins (BA 63.0%, Ka 0.209 day-1) in humans provided better accuracy than simple normalisation from monkeys. CONCLUSION: We have successfully predicted the human pharmacokinetic profiles of Fc-fusion proteins for both IV and SC administration within twofold of the observed value from monkey pharmacokinetic data by extending on reported methods for mAbs. This method will facilitate drug discovery and development of Fc-fusion proteins.

The two-domain architecture of LAMP2A regulates its interaction with Hsc70.

Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the ß-prism fold, a transmembrane domain and a short cytoplasmic tail. We previously reported the structural basis for the homophilic interaction of the lumenal domains of LAMP2A, using site-specific photo-crosslinking and/or steric hindrance within cells. In the present study, we introduced a photo-crosslinker into the cytoplasmic tail of LAMP2A and successfully detected its crosslinking with Hsc70, revealing this direct interaction for the first time. Furthermore, we demonstrated that the truncation of the membrane-distal domain within the lumenal domain of LAMP2A reduced the amount of Hsc70 that coimmunoprecipitated with LAMP2A. Our present results suggested that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with Hsc70 at the cytoplasmic surface of the lysosome.

Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells.

LAMP1 (lysosomal-associated membrane protein 1) and LAMP2 are the most abundant protein components of lysosome membranes. Both LAMPs have common structures consisting of a large lumenal domain composed of two domains (N-domain and C-domain, which are membrane-distal and -proximal, respectively), both with the ß-prism fold, a transmembrane domain, and a short cytoplasmic tail. LAMP2 is involved in various aspects of autophagy, and reportedly forms high-molecular weight complexes at the lysosomal membrane. We previously showed that LAMP2 molecules coimmunoprecipitated with each other, but whether the homophilic interaction is direct or indirect has remained to be elucidated. In the present study, we demonstrated the direct homophilic interaction of mouse LAMP2A molecules, using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces. Specifically, the results suggested that LAMP2A molecules assemble by facing each other with one side of the ß-prism (defined as side A) of the C-domains. The N-domain truncation, which increased the coimmunoprecipitation of LAMP2A molecules in our previous study, permitted the nonspecific involvement of both sides of the ß-prism (side A and side B). Thus, the presence of the N-domain restricts the LAMP2A interactions to side A-specific. The truncation of LAMP2A impaired the recruitment of GAPDH (a CMA-substrate) fused to the HaloTag protein to the surface of late endosomes/lysosomes (LE/Lys) and affected a process that generates LE/Lys. The present study revealed that the homophilic interaction of LAMP2A is direct, and the side A-specific, homophilic interaction of LAMP2A is required for the functional aspects of LAMP2A.Abbreviations: Aloc-Lys: Nε-allyloxycarbonyl-l-lysine; CMA: chaperone-mediated autophagy; FFE: free-flow electrophoresis; GAPDH-HT: glyceraldehyde-3-phosphate dehydrogenase fused to HaloTag protein; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LBPA: lysobisphosphatidic acid; LE/Lys: late endosome/lysosomes; MEFs: mouse embryonic fibroblasts; pBpa: p-benzoyl- l-phenylalanine.

A novel [5.2.1]bicyclic amine is a potent analgesic without µ opioid activity.

We identified (5R)-6-methyl-5-phenyl-1,3,4,5,6,7-hexahydro-2,5-methano-2,6-benzodiazonine (DS21980956: 4-(R)) as a novel [5.2.1]bicyclic basic compound. The scaffold was inspired by fentanyl or pethidine, which possess potent analgesic activities. DS21980956 had potent analgesic activity in the mouse acetic acid writhing test or tail flick test without agonistic activity at the µ opioid receptor (MOR). The mechanism of analgesic action of DS21980956 was considered to differ from a biased ligand, for example, TRV-130 (3, oliceridine).

Intracellular claudin-1 at the invasive front of tongue squamous cell carcinoma is associated with lymph node metastasis.

Claudins are the major component of tight junctions, which form a primary barrier to paracellular diffusion and maintain cell polarity in normal epithelia and endothelia. In cancer cells, claudins play additional roles besides serving as components of the tight junctions, and participate in anoikis or invasion. Among the claudin family proteins, claudin-1 has the most promising potential, both diagnostically and prognostically, in many types of cancers, including oral, gastric, liver, and colon cancers. However, conflicting results have been reported in relation to the degree of claudin-1 expression and the prognosis, suggesting that the expression level of claudin-1 alone is not sufficient to analyze the relationship between claudin-1 and cancer progression. As endocytic trafficking of claudin-1 has been reported in several epithelial cell types in vitro, we aimed to determine whether intracellular localization of claudin-1 is the missing aspect between claudin-1 and cancer. We investigated the expression of claudin-1 in 83 tongue squamous cell carcinoma (TSCC) pathological specimens. Although the expression level of claudin-1 based on immunohistochemistry was not associated with TSCC progression, within the high claudin-1 expression group, the incidence of intracellular localization of claudin-1 was correlated with cervical lymph node metastasis. In an in vitro experiment, claudin-1 was constitutively internalized in TSCC-derived cells. Motility of TSCC-derived cells was increased by deficiency of claudin-1, suggesting that the decrease in cell-surface claudin-1 promoted the cell migration. Therefore, intracellular localization of claudin-1 at the invasion front may represent a promising diagnostic marker of TSCC.

The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation.

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.

KIF11 as a Potential Marker of Spermatogenesis Within Mouse Seminiferous Tubule Cross-sections.

The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.

Pharmacological Modulation of Glycosphingolipid Metabolism.

The experimental approach to deplete cellular glycosphingolipids (GSLs) with the specific inhibitors of glycosphingolipid biosynthesis has the potential to identify functions of endogenous GSLs. Most GSLs are derived from glucosylceramide (GlcCer). D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibits GIcCer synthase and has been used extensively to study the biological functions of living cells. D-PDMP inhibits mTORC1 activity, which is independent of its inhibitory activity on GlcCer synthase. We also developed an analog of D-PDMP, D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-propanol (D-PBPP) lacking the effect on mTORC1. Here, we summarize the effects of D-PDMP and D-PBPP on the metabolism of GSLs and cell growth.

A Gallium-67/68-Labeled Antibody Fragment for Immuno-SPECT/PET Shows Low Renal Radioactivity Without Loss of Tumor Uptake.

Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.Experimental Design:67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab).Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images.Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309-16. ©2018 AACR.

PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum.

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER.

Characterization of Heparan Sulfate Proteoglycan-positive Recycling Endosomes Isolated from Glioma Cells.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. MATERIALS AND METHODS: Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. RESULTS: The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. CONCLUSION: Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment.

Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) assemble via distinct modes.

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) have a large, heavily glycosylated luminal domain composed of two subdomains, and are the most abundant protein components in lysosome membranes. LAMP-1 and LAMP-2 have distinct functions, and the presence of both proteins together is required for the essential regulation of autophagy to avoid embryonic lethality. However, the structural aspects of LAMP-1 and LAMP-2 have not been elucidated. In the present study, we demonstrated that the subdomains of LAMP-1 and LAMP-2 adopt the unique ß-prism fold, similar to the domain structure of the dendritic cell-specific-LAMP (DC-LAMP, LAMP-3), confirming the conserved aspect of this family of lysosome-associated membrane proteins. Furthermore, we evaluated the effects of the N-domain truncation of LAMP-1 or LAMP-2 on the assembly of LAMPs, based on immunoprecipitation experiments. We found that the N-domain of LAMP-1 is necessary, whereas that of LAMP-2 is repressive, for the organization of a multimeric assembly of LAMPs. Accordingly, the present study suggests for the first time that the assembly modes of LAMP-1 and LAMP-2 are different, which may underlie their distinct functions.

Preferential recognition of isocitrate dehydrogenase by a rabbit monoclonal antibody (ab124797) against the C-terminal peptide of RANKL.

A rabbit monoclonal antibody (Abcam ab124797), with high affinity for a synthetic peptide corresponding to the C-terminal region of the receptor activator of nuclear factor (NF)-κB ligand (RANKL), specifically recognizes a 37 kDa protein by immunoblotting, in good agreement with the molecular mass of RANKL. However, our mass spectroscopy analysis revealed that the protein recognized by the antibody is the α-subunit of NAD(+)-dependent isocitrate dehydrogenase (ICDH), a key Krebs cycle enzyme in mitochondria. Consistently, immunocytochemical staining with the antibody revealed a network organization characteristic of mitochondria, which overlapped with staining by MitoTracker and was lost after the siRNA-mediated downregulation of ICDH. The C-terminal peptide of ICDH contains similar chemical characteristics to that of the RANKL peptide and interacts with the antibody, although the affinity is a hundred times weaker. The present study provides an example of the preferential recognition of a surrogate protein by a rabbit monoclonal antibody.

Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed.

Sphingosine kinase 2 inhibitor SG-12 induces apoptosis via phosphorylation by sphingosine kinase 2.

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis.

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011bp with predicted 336 amino acids and molecular mass of 38kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494±1.536µM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4±1.2nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50=4.02±0.91µM). No regrowth of B. bovis was observed at 10µM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.