Regulation of secretory pathway kinase or kinase-like proteins in human cancers.

Secretory pathway kinase or kinase-like proteins (SPKKPs) are effective in the lumen of the endoplasmic reticulum (ER), Golgi apparatus (GA), and extracellular space. These proteins are involved in secretory signaling pathways and are distinctive from typical protein kinases. Various reports have shown that SPKKPs regulate the tumorigenesis and progression of human cancer via the phosphorylation of various substrates, which is essential in physiological and pathological processes. Emerging evidence has revealed that the expression of SPKKPs in human cancers is regulated by multiple factors. This review summarizes the current understanding of the contribution of SPKKPs in tumorigenesis and the progression of immunity. With the epidemic trend of immunotherapy, targeting SPKKPs may be a novel approach to anticancer therapy. This study briefly discusses the recent advances regarding SPKKPs.

Spotted Fever Group Rickettsia Trigger Species-Specific Alterations in Macrophage Proteome Signatures with Different Impacts in Host Innate Inflammatory Responses.

The molecular details underlying differences in pathogenicity between Rickettsia species remain to be fully understood. Evidence points to macrophage permissiveness as a key mechanism in rickettsial virulence. Different studies have shown that several rickettsial species responsible for mild forms of rickettsioses can also escape macrophage-mediated killing mechanisms and establish a replicative niche within these cells. However, their manipulative capacity with respect to host cellular processes is far from being understood. A deeper understanding of the interplay between mildly pathogenic rickettsiae and macrophages and the commonalities and specificities of host responses to infection would illuminate differences in immune evasion mechanisms and pathogenicity. We used quantitative proteomics by sequential windowed data independent acquisition of the total high-resolution mass spectra with tandem mass spectrometry (SWATH-MS/MS) to profile alterations resulting from infection of THP-1 macrophages with three mildly pathogenic rickettsiae: Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in these cells. We show that all three species trigger different proteome signatures. Our results reveal a significant impact of infection on proteins categorized as type I interferon responses, which here included several components of the retinoic acid-inducible gene I (RIG-1)-like signaling pathway, mRNA splicing, and protein translation. Moreover, significant differences in protein content between infection conditions provide evidence for species-specific induced alterations. Indeed, we confirm distinct impacts on host inflammatory responses between species during infection, demonstrating that these species trigger different levels of beta interferon (IFN-ß), differences in the bioavailability of the proinflammatory cytokine interleukin 1ß (IL-1ß), and differences in triggering of pyroptotic events. This work reveals novel aspects and exciting nuances of macrophage-Rickettsia interactions, adding additional layers of complexity between Rickettsia and host cells' constant arms race for survival. IMPORTANCE The incidence of diseases caused by Rickettsia has been increasing over the years. It has long been known that rickettsioses comprise diseases with a continuous spectrum of severity. There are highly pathogenic species causing diseases that are life threatening if untreated, others causing mild forms of the disease, and a third group for which no pathogenicity to humans has been described. These marked differences likely reflect distinct capacities for manipulation of host cell processes, with macrophage permissiveness emerging as a key virulence trait. However, what defines pathogenicity attributes among rickettsial species is far from being resolved. We demonstrate that the mildly pathogenic Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in macrophages, trigger different proteome signatures in these cells and differentially impact critical components of innate immune responses by inducing different levels of beta interferon (IFN-ß) and interleukin 1ß (IL-1ß) and different timing of pyroptotic events during infection. Our work reveals novel nuances in rickettsia-macrophage interactions, offering new clues to understand Rickettsia pathogenicity.

Comprehensive Analyses Identify APOBEC3A as a Genomic Instability-Associated Immune Prognostic Biomarker in Ovarian Cancer.

Ovarian cancer (OC) is one of the most malignant tumors whose mortality rate ranks first in gynecological tumors. Although immunotherapy sheds new light on clinical treatments, the low response still restricts its clinical use because of the unique characteristics of OC such as immunosuppressive microenvironment and unstable genomes. Further exploration on determining an efficient biomarker to predict the immunotherapy response of OC patients is of vital importance. In this study, integrative analyses were performed systematically using transcriptome profiles and somatic mutation data from The Cancer Genome Atlas (TCGA) based on the immune microenvironment and genomic instability of OC patients. Firstly, intersection analysis was conducted to identify immune-related differentially expressed genes (DEGs) and genomic instability-related DEGs. Secondly, Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3A (APOBEC3A) was recognized as a protective factor for OC, which was also verified through basic experiments such as quantitative reverse transcription PCR (RT-qPCR), immunohistochemistry (IHC), Cell Counting Kit-8 (CCK-8), and transwell assays. Thirdly, the correlation analyses of APOBEC3A expression with tumor-infiltrating immune cells (TICs), inhibitory checkpoint molecules (ICPs), Immunophenoscores (IPS), and response to anti-PD-L1 immunotherapy were further applied along with single-sample GSEA (ssGSEA), demonstrating APOBEC3A as a promising biomarker to forecast the immunotherapy response of OC patients. Last, the relationship between APOBEC3A expression with tumor mutation burden (TMB), DNA damage response (DDR) genes, and m6A-related regulators was also analyzed along with the experimental verification of immunofluorescence (IF) and RT-qPCR, comprehensively confirming the intimate association of APOBEC3A with genomic instability in OC. In conclusion, APOBEC3A was identified as a protective signature and a promising prognostic biomarker for forecasting the survival and immunotherapy effect of OC patients, which might accelerate the clinical application and improve immunotherapy effect.

Function of Protein S-Palmitoylation in Immunity and Immune-Related Diseases.

Protein S-palmitoylation is a covalent and reversible lipid modification that specifically targets cysteine residues within many eukaryotic proteins. In mammalian cells, the ubiquitous palmitoyltransferases (PATs) and serine hydrolases, including acyl protein thioesterases (APTs), catalyze the addition and removal of palmitate, respectively. The attachment of palmitoyl groups alters the membrane affinity of the substrate protein changing its subcellular localization, stability, and protein-protein interactions. Forty years of research has led to the understanding of the role of protein palmitoylation in significantly regulating protein function in a variety of biological processes. Recent global profiling of immune cells has identified a large body of S-palmitoylated immunity-associated proteins. Localization of many immune molecules to the cellular membrane is required for the proper activation of innate and adaptive immune signaling. Emerging evidence has unveiled the crucial roles that palmitoylation plays to immune function, especially in partitioning immune signaling proteins to the membrane as well as to lipid rafts. More importantly, aberrant PAT activity and fluctuations in palmitoylation levels are strongly correlated with human immunologic diseases, such as sensory incompetence or over-response to pathogens. Therefore, targeting palmitoylation is a novel therapeutic approach for treating human immunologic diseases. In this review, we discuss the role that palmitoylation plays in both immunity and immunologic diseases as well as the significant potential of targeting palmitoylation in disease treatment.

Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

Development of combination adjuvant for efficient T cell and antibody response induction against protein antigen.

Most current clinical vaccines work primarily by inducing the production of neutralizing antibodies against pathogens. Vaccine adjuvants that efficiently induce T cell responses to protein antigens need to be developed. In this study, we developed a new combination adjuvant consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D35, and an aluminum salt. Among the various combinations tested, the DOTAP/D35/aluminum salt adjuvant induced strong T cell and antibody responses against the model protein antigen with a single immunization. Adjuvant component and model antigen interaction studies in vitro also revealed that the strong mutual interactions among protein antigens and other components were one of the important factors for this efficient immune induction by the novel combination adjuvant. In addition, in vivo imaging of the antigen distribution suggested that the DOTAP component in the combination adjuvant formulation elicited transient antigen accumulation at the draining lymph nodes, possibly by antigen uptake DC migration. These results indicate the potential of the new combination adjuvant as a promising vaccine adjuvant candidate to treat infectious diseases and cancers.

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans.

Cryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

Role of Epoxide Hydrolases and Cytochrome P450s on Metabolism of KZR-616, a First-in-Class Selective Inhibitor of the Immunoproteasome.

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas.

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

The Expanding Role of Chemistry in Optimizing Proteins for Human Health Applications.

Over the past decades, therapeutics based on biological macromolecules and cells have successfully entered the clinical arena and progressively occupied an increasing share of what once was almost exclusively small molecule territory. This perspective explores the opportunities for chemists at the interface between biologics and small molecule-based products. It provides concrete examples by zooming in on the area of post-translational protein modification. The conclusion is that, rather than diminishing the relevance of chemistry in the pharmaceutical enterprise, the advent of the biologics has provided an additional playing field for synthetic and medicinal chemists, where they can contribute to the efficacy and scope of applicability of biological entities in a collaborative effort to transformatively address unmet medical needs.

Dissociation of the respiratory syncytial virus F protein-specific human IgG, IgA and IgM response.

Human respiratory syncytial virus (RSV) is one of the most important causes of severe respiratory tract infections in early childhood. The only prophylactic protection is the neutralizing antibody, palivizumab, which targets a conformational epitope of the RSV fusion (F) protein. The F protein is generated as a F0 precursor containing two furin cleavage sites allowing excision of the P27 fragment and then gives rise to a fusion-competent version consisting of the N-terminal F2 subunit and the a C-terminal F1 subunits linked by two disulphide bonds. To investigate natural human F-specific antibody responses, F2 conferring the species-specificity of RSV, was expressed in Escherichia coli. Furthermore, the F0 protein, comprising both subunits F2 and F1, was expressed as palivizumab-reactive glycoprotein in baculovirus-infected insect cells. Six overlapping F2-derived peptides lacking secondary structure were synthesized. The analysis of IgG, IgA and IgM responses of adult subjects to native versions and denatured forms of F2 and F0 and to unfolded F2-derived peptides revealed that mainly non-conformational F epitopes, some of which represented cryptic epitopes which are not exposed on the proteins were recognized. Furthermore, we found a dissociation of IgG, IgA and IgM antibody responses to F epitopes with F2 being a major target for the F-specific IgM response. The scattered and dissociated immune response to F may explain why the natural RSV-specific antibody response is only partially protective underlining the need for vaccines focusing human antibody responses towards neutralizing RSV epitopes.

Knockdown of RSAD2 attenuates B cell hyperactivity in patients with primary Sjögren's syndrome (pSS) via suppressing NF-κb signaling pathway.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is mainly characterized as abnormal activation of B cells. It is reported that radical s-adenosyl methionine domain-containing 2 (RSAD2) is overexpressed in CD19+ B cells of pSS patients, but its role in pSS B cells remains unknown. Herein, RSAD2 expression was upregulated in CD19+ B cells of pSS patients and positively correlated with the expression of interleukin-10 (IL-10) in serum. After CD40L stimulation, knockdown of RSAD2 significantly attenuated cell viability, the production levels of immunoglobins and the expression of IL-10, while promoted cell apoptosis of pSS CD19+ B cells. Mechanistically, knockdown of RSAD2 negatively regulated nuclear factor kappa-b (NF-κb) signaling pathway. In addition, overexpression of p65 prominently alleviated the inhibitory effect of RSAD2 knockdown on proliferation, immunoglobin production and IL-10 expression in CD40L-induced CD19+ B cells. Our study indicated that silencing RSAD2 attenuated pSS B cell hyperactivity via suppressing NF-κb signaling pathway, which might provide a potential therapeutic target for pSS treatment.

DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation.

Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell-engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micrometre-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR T cells, driving local tumour clearance while sparing uninjected tumours in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to substantially impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.

Recent Advances in the Development of Protein- and Peptide-Based Subunit Vaccines against Tuberculosis.

The World Health Organization (WHO) herald of the "End TB Strategy" has defined goals and targets for tuberculosis prevention, care, and control to end the global tuberculosis endemic. The emergence of drug resistance and the relative dreadful consequences in treatment outcome has led to increased awareness on immunization against Mycobacterium tuberculosis (Mtb). However, the proven limited efficacy of Bacillus Calmette-Guérin (BCG), the only licensed vaccine against Mtb, has highlighted the need for alternative vaccines. In this review, we seek to give an overview of Mtb infection and failure of BCG to control it. Afterward, we focus on the protein- and peptide-based subunit vaccine subtype, examining the advantages and drawbacks of using this design approach. Finally, we explore the features of subunit vaccine candidates currently in pre-clinical and clinical evaluation, including the antigen repertoire, the exploited adjuvanted delivery systems, as well as the spawned immune response.

Real-Time and Sensitive Immunosensor for Label-Free Detection of Specific Antigen with a Comb of Microchannel Long-Period Fiber Grating.

This study aimed to develop a comb of microchannel and immunosensor based on long-period fiber grating using the process of Lithographie Galvanoformung Abformung-like micro-electromechanical systems (LIGA-like MEMS) for real-time and label-free detection of specific antigen. The coupling between propagating core and cladding modes was conducted from the comb of microchannel long-period fiber grating (CM-LPFG). The CM-LPFG-based immunosensor consisted of a microchannel structure through photoresist stacking processes and was sandwiched with an optical fiber to obtain a long-period structure. Specific immunoglobulin against protein antigen was immobilized onto an optical fiber surface and produced a real-time resonance effect on sensing specific protein antigen from the extracted protein mixtures of the cancer cell lines. The variable transmission loss was -14.07 dB, and the resonant wavelength shift was 11.239 nm. The low limit of detection for total protein concentration was 1.363 ng/µL. Our results revealed that the CM-LPFG-based immnosensor for real-time detection of label-free protein antigen is feasible and sensitive based on the diversification of a transmission loss and achieves specific immunosensing purposes for lab-on-fiber technology.

Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Conditional Protein Rescue by Binding-Induced Protective Shielding.

Synthetic protein-level circuits offer an extra layer of cellular control on top of conventional gene-level circuits. Here, we describe a technology that allows conditional protein rescue (CPR) from proteasomal degradation using different protein inputs as masking agents. A target protein is fused to a degron tag and an affinity sensor domain. The use of nanobodies as the sensor domain offers a generalizable strategy to execute a wide range of protein-level circuits with ease. The utility of this new strategy was successfully demonstrated to distinguish cancer cells out of a healthy population using the HPV-specific E7 protein as a cellular marker. Because CPR can be programmed to execute more complex Boolean logic designs using cell-specific proteomes, this platform offers a highly modular and scalable framework for a wide range of applications based on synthetic protein circuits.