C-terminus Of Protein Research Articles

EHD1 promotes breast cancer metastasis through upregulating HIF2a expression via activating mTOR pathway.

The multistep dynamic process of metastasis is the primary cause of breast cancer deaths. C-terminal Eps15-homology domain-containing protein 1 (EHD1), a translocator associated with endocytic recycling, has been implicated in various oncogenic processes. However, the precise molecular mechanisms of EHD1-induced breast cancer metastases remain largely unexplored. Here we found that the upregulation of EHD1 in breast cancer was positively associated with distant lymph node metastasis in patients. Meanwhile, EHD1 promoted epithelial-mesenchymal transition (EMT), invasion, and metastasis of breast cancer cells in both two-dimensional (2D) and three-dimensional (3D) culture models invitro, as well as invivo. Remarkably, EHD1 can activate the AKT-mTOR pathway to upregulate the protein expression of hypoxia-inducible factor 2α (HIF2α) under normoxic conditions and subsequently enhance the invasive and metastatic breast cancer. Our findings indicated EHD1 as a new regulator of HIF2α and a potential therapeutic target for inhibiting breast cancer metastasis.

C-terminus processing in Tma12 is critical for its insecticidal activity

Tma12 is a fern-derived biopesticide (22 kDa) whose LPMO activity is associated with its insecticidal activity. The absence of the last 9 amino acids in the crystal structure of Tma12 suggested a possibility of its C-terminus processing. In this communication, we have shown the importance of protein C-terminus in the insecticidal activity. Additionally, we have also established the role of N-linked glycosylation in protein stability. Pichia produced (His)6 tagged Tma12 in two forms. The 30 kDa protein comprising 192 amino acid residues did not show insecticidal activity. Contrary, 24 kDa protein exhibited toxicity to whiteflies with an LC50 1.38 μg/ml. Absence of (His)6 tag in 24 kDa protein indicated processing at the C-terminus which was confirmed with deletion mutagenesis. Failure in expressing glycosylation defective mutant suggested the importance of glycans in the stability of Tma12. New findings together with earlier reports suggest that along with the N-terminal catalytic center, correct C-terminus is pivotal for anti-whitefly activity of Tma12.

Evolutionary Analysis and Catalytic Function of LOG Proteins in Plants

Background: The plant hormone cytokinin is a conserved regulator of plant development. LONELY GUY (LOG) proteins are pivotal in cytokinin biosynthesis. However, their origin, evolutionary history, and enzymatic characteristics remain largely uncharacterized. Methods: To elucidate LOG family evolution history and protein motif composition, we conducted phylogenetic and motif analyses encompassing representative species across the whole green plant lineage. Catalytic activity and structure analysis were conducted to thoroughly characterize the LOG proteins. Results: Our phylogeny showed that LOG proteins could be divided into five groups and revealed three major duplication events giving rise to four distinct groups of vascular LOG proteins. LOG proteins share a conserved structure characterized by a canonical motif arrangement comprising motifs 1, 2, 3, 4, 5, 6, and 7. Two significant changes in LOG motif composition occurred during the transition to land plants: the emergence of motif 3 in charophyte LOG sequences and the subsequent acquisition of motif 8 at the C-terminus of LOG proteins. Enzymatic assays demonstrated that LOG proteins can be classified into two groups based on their enzyme activity. One group act as cytokinin riboside 5′-monophosphate phosphoribohydrolase and primarily convert iPRMP to iP, while the other group act as 5′-ribonucleotide phosphohydrolase, and preferentially produce iPR from the same substrates. TaLOG5-4A1, TaLOG5-4A2, TaLOG5-5B2, and TaLOG5-D1 shared conserved residues in the critical motif and were predicted to have similar protein structures, but displayed distinct enzymatic activities. Conclusions: Our findings provide a comprehensive overview of LOG protein phylogeny and lay a foundation for further investigations into their functional diversification.

Neuropilin-1 is a co-receptor for Nerve Growth Factor-evoked pain.

Neuropilin-1 and G Alpha Interacting Protein C-terminus 1 are necessary for nerve growth factor-evoked pain and are non-opioid therapeutic targets for chronic pain.

Dichotomous intronic polyadenylation profiles reveal multifaceted gene functions in the pan-cancer transcriptome

Alternative cleavage and polyadenylation within introns (intronic APA) generate shorter mRNA isoforms; however, their physiological significance remains elusive. In this study, we developed a comprehensive workflow to analyze intronic APA profiles using the mammalian target of rapamycin (mTOR)-regulated transcriptome as a model system. Our investigation revealed two contrasting effects within the transcriptome in response to fluctuations in cellular mTOR activity: an increase in intronic APA for a subset of genes and a decrease for another subset of genes. The application of this workflow to RNA-seq data from The Cancer Genome Atlas demonstrated that this dichotomous intronic APA pattern is a consistent feature in transcriptomes across both normal tissues and various cancer types. Notably, our analyses of protein length changes resulting from intronic APA events revealed two distinct phenomena in proteome programming: a loss of functional domains due to significant changes in protein length or minimal alterations in C-terminal protein sequences within unstructured regions. Focusing on conserved intronic APA events across 10 different cancer types highlighted the prevalence of the latter cases in cancer transcriptomes, whereas the former cases were relatively enriched in normal tissue transcriptomes. These observations suggest potential, yet distinct, roles for intronic APA events during pathogenic processes and emphasize the abundance of protein isoforms with similar lengths in the cancer proteome. Furthermore, our investigation into the isoform-specific functions of JMJD6 intronic APA events supported the hypothesis that alterations in unstructured C-terminal protein regions lead to functional differences. Collectively, our findings underscore intronic APA events as a discrete molecular signature present in both normal tissues and cancer transcriptomes, highlighting the contribution of APA to the multifaceted functionality of the cancer proteome.

Serosurveillance for Plasmodium falciparum Malaria in Peruvian Army Peacekeeping Personnel, Central African Republic, 2021-2022.

Plasmodium falciparum infection threatens military populations deployed to highly malaria-endemic regions, such as Peruvian Army peacekeepers deployed to Central African Republic. During deployment, malaria cases were identified by microscopy and rapid diagnostic tests. After deployment, we performed malaria diagnosis by malachite green loop-mediated isothermal amplification and photo-induced electron transfer PCR assays. We used ELISA to test for P. falciparum C-terminal 19-kDa region merozoite surface protein 1-specific IgG from 97 peacekeepers. Malaria prevalence during deployment was 33.33% and we detected 4 cases after deployment: P. falciparum (n = 2), P. ovale (n = 1), and Plasmodium spp. (n = 1). IgG surveillance showed a seroprevalence of 31.96% in peacekeepers, who had a high P. falciparum exposure during deployment. Our findings reinforce the necessity of active surveillance in military populations to reduce the risk for introduction of new Plasmodium species and strains into the Americas from malaria-endemic areas.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76–93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min−1 mg−1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

MAVS Cys508 palmitoylation promotes its aggregation on the mitochondrial outer membrane and antiviral innate immunity

Cysteine palmitoylation or S-palmitoylation catalyzed by the ZDHHC family of acyltransferases regulates the biological function of numerous mammalian proteins as well as viral proteins. However, understanding of the role of S-palmitoylation in antiviral immunity against RNA viruses remains very limited. The adaptor protein MAVS forms functionally essential prion-like aggregates upon activation by viral RNA-sensing RIG-I-like receptors. Here, we identify that MAVS, a C-terminal tail-anchored mitochondrial outer membrane protein, is S-palmitoylated by ZDHHC7 at Cys508, a residue adjacent to the tail-anchor transmembrane helix. Using superresolution microscopy and other biochemical techniques, we found that the mitochondrial localization of MAVS at resting state mainly depends on its transmembrane tail-anchor, without regulation by Cys508 S-palmitoylation. However, upon viral infection, MAVS S-palmitoylation stabilizes its aggregation on the mitochondrial outer membrane and thus promotes subsequent propagation of antiviral signaling. We further show that inhibition of MAVS S-palmitoylation increases the host susceptibility to RNA virus infection, highlighting the importance of S-palmitoylation in the antiviral innate immunity. Also, our results indicate ZDHHC7 as a potential therapeutic target for MAVS-related autoimmune diseases.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Fragment Screening to Identify Inhibitors Targeting Ribosome Binding of Shiga Toxin 2.

Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.

Expressed Protein Ligation in Flow.

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Variants in both the N- or C-terminal domains of IHH lead to defective secretion causing short stature and skeletal defects.

Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS). To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function. In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant. IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state. These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation.

Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

#2804 Mechanisms of cAMP-induced cyst formation observed in Pkhd1-KO cells

Abstract Background and Aims Loss of ciliary protein function leads to altered epithelial properties in hereditary polycystic kidney diseases. To address molecular aspects of defective epithelial homeostasis, monolayered epithelial spheroids can be used to analyze lack of protein function and consequences of pharmacological intervention. Here, we employed epithelial cell clones deficient for fibrocystin/polyductin (FPC) protein function, the origin of ARPKD, to study the impact of FPC cytoplasmic domain constructs on cAMP-induced cystogenesis. Method We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Measurement of proportional lumen, the ratio of lumen to spheroid area, allowed detection the enhanced water and ion transport across the barrier as characteristic for secretory epithelia. Cystogenic signals were modulated by expression of the FPC C-terminal domain constructs and/or pharmacological inhibitors, and involvement of ion channels was addressed. Results In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. The rise in proportional lumen was sensitive to inhibition of chloride-channels, CFTR and TMEM16A, and Src kinase. Fsk-mediated cyst induction led to activation and increased Y705 phosphorylation of STAT3. To address the impact of FPC on cystogenic signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed Fsk-induced cyst formation. In addition, the FPC domain reduced activation of Src and STAT3, leading to lower STAT3-dependent transcription. Conclusion In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain, leading to a gain-of-function, suppresses Src and STAT3 activity in Pkhd1-KO epithelia and limits cyst growth in vitro. Control of cystogenic Src/STAT3 signaling can be considered a physiologic function of the FPC protein.

Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary β subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

A Small Epitope Tagging on the C-Terminus of a Target Protein Requires Extra Amino Acids to Enhance the Immune Responses of the Corresponding Antibody.

Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are unavailable, protein tagging is a useful alternative. Small epitope tags, typically less than 10 amino acids, are widely used in protein research due to the simple modification through PCR and reduced impact on the target protein's function compared to larger tags. The 2B8 epitope tag (RDPLPFFPP), reported by us in a previous study, has high specificity and sensitivity to the corresponding antibody. However, when attached to the C-terminus of the target protein in immunoprecipitation experiments, we observed a decrease in detection signal with reduced immunity and low protein recovery. This phenomenon was not unique to 2B8 and was also observed with the commercially available Myc tag. Our study revealed that C-terminal tagging of small epitope tags requires the addition of more than one extra amino acid to enhance (restore) antibody immunities. Moreover, among the amino acids we tested, serine was the best for the 2B8 tag. Our findings demonstrated that the interaction between a small epitope and a corresponding paratope of an antibody requires an extra amino acid at the C-terminus of the epitope. This result is important for researchers planning studies on target proteins using small epitope tags.

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.