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The multifaceted chemo-immune resistance is the principal barrier to achieving cure in cancer patients. Identifying a target that is critically involved in chemo-immune-resistance represents an attractive strategy to improve cancer treatment. iRhom1 plays a role in cancer cell proliferation and its expression is negatively correlated with immune cell infiltration. Here we show that iRhom1 decreases chemotherapy sensitivity by regulating the MAPK14-HSP27 axis. In addition, iRhom1 inhibits the cytotoxic T-cell response by reducing the stability of ERAP1 protein and the ERAP1-mediated antigen processing and presentation. To facilitate the therapeutic translation of these findings, we develop a biodegradable nanocarrier that is effective in codelivery of iRhom pre-siRNA (pre-siiRhom) and chemotherapeutic drugs. This nanocarrier is effective in tumor targeting and penetration through both enhanced permeability and retention effect and CD44-mediated transcytosis in tumor endothelial cells as well as tumor cells. Inhibition of iRhom1 further facilitates tumor targeting and uptake through inhibition of CD44 cleavage. Co-delivery of pre-siiRhom and a chemotherapy agent leads to enhanced antitumor efficacy and activated tumor immune microenvironment in multiple cancer models in female mice. Targeting iRhom1 together with chemotherapy could represent a strategy to overcome chemo-immune resistance in cancer treatment.

The nomenclature of fungi has undergone significant changes in the last decade. Renaming of fungi has always occurred and will continue, largely due to the influence of molecular methods in taxonomy, diagnosis, and epidemiology. Recently, advancements in molecular techniques and genetic analysis have have led to changes in the classification of dermatophytes that could potentially affect the clinical diagnosis and management of the disease. Fungal culture is still considered the "gold standard" for the diagnosing dermatophytosis; however, modern molecular assays have overcome the major drawbacks of culture and can be used in conjunction with it. Dermatophytes are one of the oldest groups of microorganisms known to cause disease in humans and animals. The ability of dermatophytes to secrete keratinolytic enzymes in vitro has led to the study of secretory proteases. Studies on the quantity and function of keratinolytic proteases produced by dermatophytes are increasing, and the enzymatic properties of these compounds have been investigated. Because of the importance of this group of fungi in public health, this review focuses on the new classification of dermatophytes, including the renaming of important medical species, as well as the pathogenic factors associated with them. The search strategy used was a narrative review, using national and international sources such as PubMed, Web of Science, Google Scholar, Scopus, and the country's periodicals database to collect information. In the proposed new classification, Arthroderma includes 21 species, Trichophyton 16, Nanzia 9, Microsporum 3, Paraphyton 3, Epidermophyton and Lophophyton one each. However, more detailed studies are needed to determine the boundaries of these species. Based on previous studies, it can be concluded that the current classification of common anthropophilic dermatophytes is fairly stable and is unlikely to change drastically in the future. While the study of geophilic dermatophytes has been inadequate compared to many zoophilic species, further taxonomic innovation in these groups is expected. In addition, research into the pathogenic mechanisms of dermatophytes has shown that endopeptidases and exopeptidases play an important role in the pathogenesis of dermatophytes by degrading keratin and converting it into free peptides and smaller amino acids. The most important endopeptidases are from the fungilysin family (M36) and two genes encoding MEP1 and MEP5, while the most important extracellular aminopeptidases are the leucine aminopeptidases Lap1 and Lap2 (M28A family) and the dipeptidyl peptidases DppIV and DppV, which are considered to be dermatophyte exopeptidases. The results of previous studies suggest that by identifying these major proteases, it is possible to isolate and identify dermatophyte species. Based on the results of this study, many factors related to the classification and pathogenesis of dermatophytes remain inadequately expressed in existing research. However, the factors discussed in this study serve as a guide for the identification of relevant factors. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of acute kidney injury (AKI) relies on serum creatinine (SCr) changes. This study investigated if urinary aminopeptidases are early and predictive biomarkers of cardiac surgery-associated AKI (CSA-AKI). Methods: Glutamyl aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), proteinuria, albuminuria, N-acetyl-β-D-glucosaminidase (NAG), and neutrophile gelatinase-associated lipocalin (NGAL) were measured in urine samples from 44 patients at arrival in the intensive care unit (ICU) after cardiac surgery. Sensitivity, specificity, and positive and negative predictive value for diagnosis of stages 1, 2, and 3 of AKI were analyzed for the highest quartile of each marker. We also studied the relationship with SCr after surgery, 6- and 12-month glomerular filtration rates (GFRs), and other long-term events over the next 5 years. Results: GluAp diagnosed the maximal number of patients that developed stage 2 or 3 of AKI, increasing diagnostic sensitivity from 0% to 75%. In addition, GluAp and DPP4 were related to the decrease in GFR at 6 or 12 months after surgery. Conclusions: Urinary aminopeptidases are a potential tool for the early diagnosis of CSA-AKI, with GluAp being the most effective marker for diagnosing stage 2 or 3 of AKI at ICU admission. GluAp and DPP4 serve as predictive biomarkers for a decrease in GFR. [ABSTRACT FROM AUTHOR]

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis. How methionine excision and acetylation are co-translationally coordinated at the ribosome has remained elusive. Here, the authors report cryo-EM structures of two different multi-protein assemblies capable of both successive enzymatic functions. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

Diabetic kidney disease (DKD) is recognized as an important public health challenge. However, its genomic mechanisms are poorly understood. To identify rare variants for DKD, we conducted a whole-exome sequencing (WES) study leveraging large cohorts well-phenotyped for chronic kidney disease and diabetes. Our two-stage WES study included 4372 European and African ancestry participants from the Chronic Renal Insufficiency Cohort and Atherosclerosis Risk in Communities studies (stage 1) and 11 487 multi-ancestry Trans-Omics for Precision Medicine participants (stage 2). Generalized linear mixed models, which accounted for genetic relatedness and adjusted for age, sex and ancestry, were used to test associations between single variants and DKD. Gene-based aggregate rare variant analyses were conducted using an optimized sequence kernel association test implemented within our mixed model framework. We identified four novel exome-wide significant DKD-related loci through initiating diabetes. In single-variant analyses, participants carrying a rare, in-frame insertion in the DIS3L2 gene (rs141560952) exhibited a 193-fold increased odds [95% confidence interval (CI): 33.6, 1105] of DKD compared with noncarriers (P = 3.59 × 10-9). Likewise, each copy of a low-frequency KRT6B splice-site variant (rs425827) conferred a 5.31-fold higher odds (95% CI: 3.06, 9.21) of DKD (P = 2.72 × 10-9). Aggregate gene-based analyses further identified ERAP2 (P = 4.03 × 10-8) and NPEPPS (P = 1.51 × 10-7), which are both expressed in the kidney and implicated in renin-angiotensin-aldosterone system modulated immune response. In the largest WES study of DKD, we identified novel rare variant loci attaining exome-wide significance. These findings provide new insights into the molecular mechanisms underlying DKD.

Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.

Abstract CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

Aminopeptidases are a group of enzymatic proteins crucial for protein digestion, catalyzing the cleavage of amino acids at the N-terminus of peptides. Among them are ERAP1 (coding for endoplasmic reticulum aminopeptidase 1), ERAP2 (coding for endoplasmic reticulum aminopeptidase 2), and LNPEP (coding for leucyl and cystinyl aminopeptidase). These genes encoding these enzymes are contiguous and located on the same chromosome (5q21); they share structural homology and functions and are associated with immune-mediated diseases. These aminopeptidases play a key role in immune pathology by cleaving peptides to optimal sizes for binding to the major histocompatibility complex (MHC) and contribute to cellular homeostasis. By their ability to remove the extracellular region of interleukin 2 and 6 receptors (IL2, IL6) and the tumor necrosis factor receptor (TNF), ERAP1 and ERAP2 are involved in regulating the innate immune response and, finally, in blood pressure control and angiogenesis. The combination of specific genetic variations in these genes has been linked to various conditions, including autoimmune and autoinflammatory diseases and cancer, as well as hematological and dermatological disorders. This literature review aims to primarily explore the impact of ERAP1 polymorphisms on its enzymatic activity and function. Through a systematic examination of the available literature, this review seeks to provide valuable insights into the role of ERAP1 in the pathogenesis of various diseases and its potential implications for targeted therapeutic interventions. Through an exploration of the complex interplay between ERAP1 and various disease states, this review contributes to the synthesis of current biomedical research findings and their implications for personalized medicine.

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells. [ABSTRACT FROM AUTHOR]

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

Excision of the initiator methionine is among the first co-translational processes that occur at the ribosome. While this crucial step in protein maturation is executed by two types of methionine aminopeptidases in eukaryotes (MAP1 and MAP2), additional roles in disease and translational regulation have drawn more attention to MAP2. Here, we report several cryo-EM structures of human and fungal MAP2 at the 80S ribosome. Irrespective of nascent chains, MAP2 can occupy the tunnel exit. On nascent chain displaying ribosomes, the MAP2-80S interaction is highly dynamic and the MAP2-specific N-terminal extension engages in stabilizing interactions with the long rRNA expansion segment ES27L. Loss of this extension by autoproteolytic cleavage impedes interactions at the tunnel, while promoting MAP2 to enter the ribosomal A-site, where it engages with crucial functional centers of translation. These findings reveal that proteolytic remodeling of MAP2 severely affects ribosome binding, and set the stage for targeted functional studies. The role of methionine aminopeptidase 2 (MAP2) at the ribosome goes beyond N-terminal methionine excision. Klein et al. use cryo-EM to identify a second MAP2 binding site on the ribosome, and describe the dynamic interactions of MAP2 at the ribosome. [ABSTRACT FROM AUTHOR]

It has been suggested that the neuro-visceral integration works asymmetrically and that this asymmetry is dynamic and modifiable by physio-pathological influences. Aminopeptidases of the renin–angiotensin system (angiotensinases) have been shown to be modifiable under such conditions. This article analyzes the interactions of these angiotensinases between the left or right frontal cortex (FC) and the same enzymes in the hypothalamus (HT), pituitary (PT), adrenal (AD) axis (HPA) in control spontaneously hypertensive rats (SHR), in SHR treated with a hypotensive agent in the form of captopril (an angiotensin-converting enzyme inhibitor), and in SHR treated with a hypertensive agent in the form of the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). In the control SHR, there were significant negative correlations between the right FC with HPA and positive correlations between the left FC and HPA. In the captopril group, the predominance of negative correlations between the right FC and HPA and positive correlations between the HPA and left FC was maintained. In the L-NAME group, a radical change in all types of interactions was observed; particularly, there was an inversion in the predominance of negative correlations between the HPA and left FC. These results indicated a better balance of neuro-visceral interactions after captopril treatment and an increase in these interactions in the hypertensive animals, especially in those treated with L-NAME. [ABSTRACT FROM AUTHOR]

Aminopeptidases catalyze the hydrolysis of peptides into constituent amino acids and are therefore a key step in nitrogen (N) depolymerization and downstream mineralization. Differences in carbon (C):N as well as sulfur (S) content of amino acids present the possibility of amino acid-specific aminopeptidase activities revealing distinct information on N and non-N processes in soils. However, chromogenic assays of soil aminopeptidases using para-nitroaniline (pNA) substrates are underutilized and have largely focused on just two aminopeptidases to-date. We evaluated activities of eight amino acid-specific aminopeptidases using pNA substrates for arginine (ARG), glycine (GLY), alanine (ALA), lysine (LYS), glutamic acid (GLU), methionine (MET), proline (PRO) and leucine (LEU) aminopeptidases in a high-resolution sampling (30 samples per 0.01 ha−1 treatment plot) of nine 145-year treatment plots resulting from a factorial combination of three crop rotations and three fertility treatments on a Argiudoll in North Central United States. Across treatments, we identified non-detectable activities of ARG and PRO, and detectable activities of GLY, ALA, LYS, GLU, MET, and LEU ranging 0.2 to 400 nmol pNA h−1 g−1. Except for LYS and GLU activities, aminopeptidase activities were homogeneously distributed within treatments (n=30 locations per plot), and exhibited a mean coefficient of variation (CV) of 9 to 30 % and spatial range of 4.1 to 9.0 m. Activities of ALA and MET had the lowest CV and greatest contribution in explaining overall variability of aminopeptidase activities and were more sensitive to treatments compared to GLY and LEU activity. Multivariate analysis revealed a positive correlation among aminopeptidase activities, with major variation explained by β-glucosidase activity followed by pH and total N (TN). Absolute aminopeptidase activities generally reflected soil organic C (SOC) and TN concentrations, with greater activities under diverse rotation with organic fertilization (manure) and lower activities under continuous maize without fertilization. However, aminopeptidase activities normalized to SOC concentration were greater in soils without fertilization relative to organic fertilization. This study provides benchmark values of soil aminopeptidase activities assayed by pNA substrates using the world’s second oldest continuous agricultural experiment, including six aminopeptidases that have not been previously assayed with these substrates. Given their low magnitude of variability, high spatial correlation, and sensitivity to management, assays of aminopeptidase activities – particularly MET and ALA activities – could be fruitful tools for better understanding soil N cycling. Future work should evaluate assay optimization and expand to additional amino acid-specific substrates using pNA-based substrates.

Background: The resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain. Methods: The activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined. Results: Enzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters. Conclusions: Our dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that deletion of an intracellular leucine aminopeptidase results in attenuated virulence of S. aureus. Herein we explore the role of 10 other aminopeptidases in S. aureus pathogenesis. Using a human blood survival assay we identified mutations in two enzymes from the M20B family (PepT1 and PepT2) as having markedly decreased survival compared to the parent. We further reveal that pepT1, pepT2 and pepT1/2 mutant strains are impaired in their ability to resist phagocytosis by, and engender survival within, human macrophages. Using a co-infection model of murine sepsis, we demonstrate impairment of dissemination and survival for both single mutants that is even more pronounced in the double mutant. We show that these enzymes are localized to the cytosol and membrane but are not necessary for peptide-based nutrition, a hallmark of cell-associated aminopeptidases. Furthermore, none of the survival defects appear to be the result of altered virulence factor production. An exploration of their regulation reveals that both are controlled by known regulators of the S. aureus virulence process, including Agr, Rot and/or SarA, and that this cascade may be mediated by FarR. Structural modeling of PepT1 reveals it bears all the hallmarks of a tripeptidase, whilst PepT2 differs significantly in its catalytic pocket, suggesting a broader substrate preference. In sum, we have identified two M20B aminopeptidases that are integral to S. aureus pathogenesis. The future identification of protein and/or peptide targets for these proteases will be critical to understanding their important virulence impacting functions. [ABSTRACT FROM AUTHOR]

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal-dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn2+ and Mn2+ , while the second structure has two Zn2+ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N-terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C-terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.

Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well as peptides released from the proteasome, including polyglutamine. We report the crystal structure of PSA at 2.3 Ǻ. Overall, the enzyme adopts a V-shaped architecture with four domains characteristic of the M1 family aminopeptidases, but it is in a less compact conformation compared to most M1 enzymes of known structure. A microtubule binding sequence is present in a C-terminal HEAT repeat domain of the enzyme in a position where it might serve to mediate interaction with tubulin. In the catalytic metallopeptidase domain, an elongated active site groove lined with aromatic and hydrophobic residues and a large S1 subsite may play a role in broad substrate recognition. The structure with bound polyglutamine shows a possible interacting mode of this peptide, which is supported by mutation. [ABSTRACT FROM AUTHOR]

Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

Cotranslational modification of the nascent polypeptide chain is one of the first events during the birth of a new protein. In eukaryotes, methionine aminopeptidases (MetAPs) cleave off the starter methionine, whereas N-acetyl-transferases (NATs) catalyze N-terminal acetylation. MetAPs and NATs compete with other cotranslationally acting chaperones, such as ribosome-associated complex (RAC), protein targeting and translocation factors (SRP and Sec61) for binding sites at the ribosomal tunnel exit. Yet, whereas well-resolved structures for ribosome-bound RAC, SRP and Sec61, are available, structural information on the mode of ribosome interaction of eukaryotic MetAPs or of the five cotranslationally active NATs is only available for NatA. Here, we present cryo-EM structures of yeast Map1 and NatB bound to ribosome-nascent chain complexes. Map1 is mainly associated with the dynamic rRNA expansion segment ES27a, thereby kept at an ideal position below the tunnel exit to act on the emerging substrate nascent chain. For NatB, we observe two copies of the NatB complex. NatB-1 binds directly below the tunnel exit, again involving ES27a, and NatB-2 is located below the second universal adapter site (eL31 and uL22). The binding mode of the two NatB complexes on the ribosome differs but overlaps with that of NatA and Map1, implying that NatB binds exclusively to the tunnel exit. We further observe that ES27a adopts distinct conformations when bound to NatA, NatB, or Map1, together suggesting a contribution to the coordination of a sequential activity of these factors on the emerging nascent chain at the ribosomal exit tunnel. Cryo-EM structures of yeast methionine aminopeptidase and N-acetyltransferase B in complex with ribosomes shed light on the involvement of rRNA expansion segment ES27a in orchestrating modifications of newly emerging polypeptide chains during translation. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord, brain stem, and cerebral cortex. Biomarkers for ALS are essential for disease detection and to provide information on potential therapeutic targets. Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or substrates such as neuropeptides. Since certain aminopeptidases are known to increase the risk of neurodegeneration, such mechanisms may reveal new targets to determine their association with ALS risk and their interest as a diagnostic biomarker. The authors performed a systematic review and meta-analyses of genome-wide association studies (GWASs) to identify reported aminopeptidases genetic loci associated with the risk of ALS. PubMed, Scopus, CINAHL, ISI Web of Science, ProQuest, LILACS, and Cochrane databases were searched to retrieve eligible studies in English or Spanish, published up to 27 January 2023. A total of 16 studies were included in this systematic review, where a series of aminopeptidases could be related to ALS and could be promising biomarkers (DPP1, DPP2, DPP4, LeuAP, pGluAP, and PSA/NPEPPS). The literature reported the association of single-nucleotide polymorphisms (SNPs: rs10260404 and rs17174381) with the risk of ALS. The genetic variation rs10260404 in the DPP6 gene was identified to be highly associated with ALS susceptibility, but meta-analyses of genotypes in five studies in a matched cohort of different ancestry (1873 cases and 1861 control subjects) showed no ALS risk association. Meta-analyses of eight studies for minor allele frequency (MAF) also found no ALS association for the "C" allele. The systematic review identified aminopeptidases as possible biomarkers. However, the meta-analyses for rs1060404 of DPP6 do not show a risk associated with ALS. [ABSTRACT FROM AUTHOR]

A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and Pep X) activities, were evaluated. Most of the strains demonstrated salt tolerance to 6% of NaCl, while only two L. delbruekii (P14, P38), one L. rhamnosus (P50) and one L. plantarum (Q3C4) were able to grow in the presence of 10% (w/v) of NaCl. Except for 2 L. plantarum (Q1C6 and Q3C4), all strains showed low or medium acidifying activity and good proteolytic features. Furthermore, lipolytic activity was revealed in none of the strains, while the production of EPS and diacetyl was widespread and variable among the tested strains. Finally, regarding aminopeptidase activities, 1 L. delbrueckii (P10), 1 L. rhamnosus (P50), and 1 L. lactis (Q5C6) were considered as the better performing, showing high values of both Pep N and Pep X. Based on data presented here, the aforementioned strains could be suggested as promising adjunct cultures in cheesemaking. [ABSTRACT FROM AUTHOR]

This study involves the in-vitro and in-vivo anti-TB potency and in-vivo safety of Transitmycin (TR) (PubChem CID:90659753)- identified to be a novel secondary metabolite derived from Streptomyces sp (R2). TR was tested in-vitro against drug resistant TB clinical isolates (n = 49). 94% of DR-TB strains (n = 49) were inhibited by TR at 10μg ml-1. In-vivo safety and efficacy studies showed that 0.005mg kg-1 of TR is toxic to mice, rats and guinea pigs, while 0.001mg kg-1 is safe, infection load did not reduce. TR is a potent DNA intercalator and also targets RecA and methionine aminopeptidases of Mycobacterium. Analogue 47 of TR was designed using in-silico based molecule detoxification approaches and SAR analysis. The multiple targeting nature of the TR brightens the chances of the analogues of TR to be a potent TB therapeutic molecule even though the parental compound is toxic. Analog 47 of TR is proposed to have non-DNA intercalating property and lesser in-vivo toxicity with high functional potency. This study attempts to develop a novel anti-TB molecule from microbial sources. Though the parental compound is toxic, its analogs are designed to be safe through in-silico approaches. However, further laboratory validations on this claim need to be carried out before labelling it as a promising anti-TB molecule. [ABSTRACT FROM AUTHOR]

Birdshot Uveitis (Birdshot) is a rare eye condition that affects HLA-A29-positive individuals and could be considered a prototypic member of the recently proposed 'MHC-I (major histocompatibility complex class I)-opathy' family. Genetic studies have pinpointed the endoplasmic reticulum aminopeptidase (ERAP1) and (ERAP2) genes as shared associations across MHC-I-opathies, which suggests ERAP dysfunction may be a root cause for MHC-I-opathies. We mapped the ERAP1 and ERAP2 haplotypes in 84 Dutch cases and 890 controls. We identified association at variant rs10044354, which mediated a marked increase in ERAP2 expression. We also identified and cloned an independently associated ERAP1 haplotype (tagged by rs2287987) present in more than half of the cases; this ERAP1 haplotype is also the primary risk and protective haplotype for other MHC-I-opathies. We show that the risk ERAP1 haplotype conferred significantly altered expression of ERAP1 isoforms in transcriptomic data (n = 360), resulting in lowered protein expression and distinct enzymatic activity. Both the association for rs10044354 (meta-analysis: odds ratio (OR) [95% CI]=2.07[1.58-2.71], P = 1.24 × 10(-7)) and rs2287987 (OR[95% CI]: =2.01[1.51-2.67], P = 1.41 × 10(-6)) replicated and showed consistent direction of effect in an independent Spanish cohort of 46 cases and 2103 controls. In both cohorts, the combined rs2287987-rs10044354 haplotype associated with Birdshot more strongly than either variant alone [meta-analysis: P=3.9 × 10(-9)]. Finally, we observed that ERAP2 protein expression is dependent on the ERAP1 background across three European populations (n = 3353). In conclusion, a functionally distinct combination of ERAP1 and ERAP2 are a hallmark of Birdshot and provide rationale for strategies designed to correct ERAP function for treatment of Birdshot and MHC-I-opathies more broadly.

While genetic variants are known to be associated with overall gene abundance in stimulated immune cells, less is known about their effects on alternative isoform usage. By analyzing RNA-seq profiles of monocyte-derived dendritic cells from 243 individuals, we uncovered thousands of unannotated isoforms synthesized in response to influenza infection and type 1 interferon stimulation. We identified more than a thousand quantitative trait loci (QTLs) associated with alternate isoform usage (isoQTLs), many of which are independent of expression QTLs (eQTLs) for the same gene. Compared with eQTLs, isoQTLs are enriched for splice sites and untranslated regions, but depleted of sequences upstream of annotated transcription start sites. Both eQTLs and isoQTLs explain a significant proportion of the disease heritability attributed to common genetic variants. At the ERAP2 locus, we shed light on the function of the gene and how two frequent, highly differentiated haplotypes with intermediate frequencies could be maintained by balancing selection. At baseline and following type 1 interferon stimulation, the major haplotype is associated with low ERAP2 expression caused by nonsense-mediated decay, while the minor haplotype, known to increase Crohn's disease risk, is associated with high ERAP2 expression. In response to influenza infection, we found two uncharacterized isoforms expressed from the major haplotype, likely the result of multiple perfectly linked variants affecting the transcription and splicing at the locus. Thus, genetic variants at a single locus could modulate independent gene regulatory processes in innate immune responses and, in the case of ERAP2, may confer a historical fitness advantage in response to virus.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Background: COVID-19 outcomes display multiple unexpected varieties, ranging from unnoticed symptomless infection to death, without any previous alarm or known aggravating factors. Aim: To appraise the impact of ACErs4291(A/T) and ERAP1rs26618(T/C) human polymorphisms on the outcome of COVID-19. Subjects and methods: In total, 240 individuals were enrolled in the study (80 with severe manifestations, 80 with mild manifestations, and 80 healthy persons). ACErs4291(A/T) and ERAP1rs26618(T/C) genotyping was performed using RT-PCR. Results: The frequency of the ACErs4291AA genotype was higher among the severe COVID-19 group than others (p < 0.001). The ERAP1rs26618TT genotype frequency was higher among the severe COVID-19 group in comparison with the mild group (p < 0.001) and non-infected controls (p = 0.0006). The frequency of the ACErs4291A allele was higher among severe COVID-19 than mild and non-infected groups (64.4% vs. 37.5%, and 34.4%, respectively), and the ERAP1rs26618T allele was also higher in the severe group (67.5% vs. 39.4%, and 49.4%). There was a statistically significant association between severe COVID-19 and ACErs4291A or ERAP1rs26618T alleles. The coexistence of ACErs4291A and ERAP1rs26618T alleles in the same individual increase the severity of the COVID-19 risk by seven times [OR (95%CI) (LL–UL) = 7.058 (3.752–13.277), p < 0.001). A logistic regression analysis revealed that age, male gender, non-vaccination, ACErs4291A, and ERAP1rs26618T alleles are independent risk factors for severe COVID-19. Conclusions: Persons carrying ACErs4291A and/or ERAP1rs26618T alleles are at higher risk of developing severe COVID-19. [ABSTRACT FROM AUTHOR]

Ophiocordyceps australis (Ascomycota, Hypocreales, Ophiocordycipitaceae) is a classic entomopathogenic fungus that parasitizes ants (Hymenoptera, Ponerinae, Ponerini). Nonetheless, according to our results, this fungal species also exhibits a complete set of genes coding for plant cell wall degrading Carbohydrate-Active enZymes (CAZymes), enabling a full endophytic stage and, consequently, its dual ability to both parasitize insects and live inside plant tissue. The main objective of our study was the sequencing and full characterization of the genome of the fungal strain of O. australis (CCMB661) and its predicted secretome. The assembled genome had a total length of 30.31 Mb, N50 of 92.624 bp, GC content of 46.36%, and 8,043 protein-coding genes, 175 of which encoded CAZymes. In addition, the primary genes encoding proteins and critical enzymes during the infection process and those responsible for the host–pathogen interaction have been identified, including proteases (Pr1, Pr4), aminopeptidases, chitinases (Cht2), adhesins, lectins, lipases, and behavioral manipulators, such as enterotoxins, Protein Tyrosine Phosphatases (PTPs), and Glycoside Hydrolases (GHs). Our findings indicate that the presence of genes coding for Mad2 and GHs in O. australis may facilitate the infection process in plants, suggesting interkingdom colonization. Furthermore, our study elucidated the pathogenicity mechanisms for this Ophiocordyceps species, which still is scarcely studied. [ABSTRACT FROM AUTHOR]

Background: Despite growing knowledge regarding the pathogenesis of autoimmune diseases (ADs) onset, the current treatment remains unsatisfactory. This study aimed to identify innovative therapeutic targets for ADs through various analytical approaches., Research Design and Methods: Utilizing Mendelian randomization, Bayesian co-localization, phenotype scanning, and protein-protein interaction network, we explored potential therapeutic targets for 14 ADs and externally validated our preliminary findings., Results: This study identified 12 circulating proteins as potential therapeutic targets for six ADs. Specifically, IL12B was judged to be a risk factor for ankylosing spondylitis ( p  = 1.61E - 07). TYMP ( p  = 6.28E - 06) was identified as a protective factor for ulcerative colitis. For Crohn's disease, ERAP2 ( p  = 4.47E - 14), HP ( p  = 2.08E - 05), and RSPO3 ( p  = 6.52E - 07), were identified as facilitators, whereas FLRT3 ( p  = 3.42E - 07) had a protective effect. In rheumatoid arthritis, SWAP70 ( p  = 3.26E - 10), SIGLEC6 ( p  = 2.47E - 05), ISG15 ( p  = 3.69E - 05), and FCRL3 ( p  = 1.10E - 10) were identified as risk factors. B4GALT1 ( p  = 6.59E - 05) was associated with a lower risk of Type 1 diabetes (T1D). Interestingly, CTSH was identified as a protective factor for narcolepsy ( p  = 1.58E - 09) but a risk factor for T1D ( p  = 7.36E - 11), respectively. External validation supported the associations of eight of these proteins with three ADs., Conclusions: Our integrated study identified 12 potential therapeutic targets for ADs and provided novel insights into future drug development for ADs.

Brain dopamine, in relation to the limbic system, is involved in cognition and emotion. These functions are asymmetrically processed. Hypertension not only alters such functions but also their asymmetric brain pattern as well as their bilateral pattern of neurovisceral integration. The central and peripheral renin-angiotensin systems, particularly the aminopeptidases involved in its enzymatic cascade, play an important role in blood pressure control. In the present study, we report how these aminopeptidases from left and right cortico-limbic locations, plasma and systolic blood pressure interact among them in spontaneously hypertensive rats (SHR) unilaterally depleted of dopamine. The study comprises left and right sham and left and right lesioned (dopamine-depleted) rats as research groups. Results revealed important differences in the bilateral behavior comparing sham left versus sham right, lesioned left versus lesioned right, and sham versus lesioned animals. Results also suggest an important role for the asymmetrical functioning of the amygdala in cardiovascular control and an asymmetrical behavior in the interaction between the medial prefrontal cortex, hippocampus and amygdala with plasma, depending on the left or right depletion of dopamine. Compared with previous results of a similar study in Wistar-Kyoto (WKY) normotensive rats, the asymmetrical behaviors differ significantly between both WKY and SHR strains. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate the effect of dried apple pomace (DAP) as a feed additive on the enzymatic activity and non-enzymatic compounds belonging to the antioxidant system in cattle rumen fluid. The experiment included 4 Polish Holstein–Friesian cannulated dairy cows and lasted 52 days. The control group was fed with the standard diet, while in the experimental group, 6% of the feedstuff was replaced by dried apple pomace. After the feeding period, ruminal fluid was collected. The spectrophotometric technique for the activity of lysosomal enzymes, the content of vitamin C, polyphenols, and the potential to scavenge the free DPPH radical was used. The enzyme immunoassay tests (ELISA) were used to establish the activity of antioxidants enzymes and MDA. Among the rumen aminopeptidases, a significant reduction (p < 0.01) from 164.00 to 142.00 was observed for leucyl-aminopeptidase. The activity of glycosidases was decreased for HEX (from 231.00 to 194.00) and β-Glu (from 1294.00 to 1136.00), while a significant statistically increase was noticed for BGRD (from 31.10 to 42.40), α-Glu (from 245.00 to 327.00), and MAN (from 29.70 to 36.70). Furthermore, the activity of catalase and GSH (p < 0.01) was inhibited. In turn, the level of vitamin C (from 22.90 to 24.10) and MDA (from 0.36 to 0.45) was statistically higher (p < 0.01). The most positive correlations were observed between AlaAP and LeuAP (r = 0.897) in the aminopeptidases group and between β-Gal and MAN (r = 0.880) in the glycosidases group. Furthermore, one of the most significant correlations were perceived between SOD and AlaAP (r = 0.505) and AcP (r = 0.450). The most negative correlation was noticed between α-Gal and DPPH (r = −0.533) based on these observations. Apple pomace as a feed additive has an influence on lysosomal degradation processes and modifies oxidation–reduction potential in the rumen fluid. Polyphenols and other low-weight antioxidant compounds are sufficient to maintain redox balance in the rumen. [ABSTRACT FROM AUTHOR]

AimsThere are no treatments for the extreme hyperphagia and obesity in Prader-Willi syndrome (PWS). The bestPWS clinical trial assessed the efficacy, safety and tolerability of the methionine aminopeptidase 2 (MetAP2) inhibitor, beloranib.Materials and methodsParticipants with PWS (12-65 years old) were randomly assigned (1:1:1) to biweekly placebo, 1.8 mg beloranib or 2.4 mg beloranib injection for 26 weeks at 15 US sites. Co-primary endpoints were the changes in hyperphagia [measured by Hyperphagia Questionnaire for Clinical Trials (HQ-CT); possible score 0-36] and weight by intention-to-treat. ClinicalTrials.gov registration: NCT02179151.ResultsOne-hundred and seven participants were included in the intention-to-treat analysis: placebo (n = 34); 1.8 mg beloranib (n = 36); or 2.4 mg beloranib (n = 37). Improvement (reduction) in HQ-CT total score was greater in the 1.8 mg (mean difference -6.3, 95% CI -9.6 to -3.0; P = .0003) and 2.4 mg beloranib groups (-7.0, 95% CI -10.5 to -3.6; P = .0001) vs placebo. Compared with placebo, weight change was greater with 1.8 mg (mean difference - 8.2%, 95% CI -10.8 to -5.6; P < .0001) and 2.4 mg beloranib (-9.5%, 95% CI -12.1 to -6.8; P < .0001). Injection site bruising was the most frequent adverse event with beloranib. Dosing was stopped early due to an imbalance in venous thrombotic events in beloranib-treated participants (2 fatal events of pulmonary embolism and 2 events of deep vein thrombosis) compared with placebo.ConclusionsMetAP2 inhibition with beloranib produced statistically significant and clinically meaningful improvements in hyperphagia-related behaviours and weight loss in participants with PWS. Although investigation of beloranib has ceased, inhibition of MetAP2 is a novel mechanism for treating hyperphagia and obesity.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

Background: The age-related increase in blood pressure in spontaneously hypertensive rats (SHRs) is associated to cardiac hypertrophy, heart failure, and renal injury. Here, we investigated for the first time the urinary enzymatic activities of glutamil aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), and Klotho urinary levels, proteins that are strongly expressed in the kidney, as early biomarkers of renal injury in SHRs. Methods: Male SHR and Wistar Kyoto (WKY) rats were studied from 2 to 8 months old. Systolic blood pressure (SBP), the heart rate (HR), metabolic variables, and urinary markers were measured monthly. At the end of the study, a histopathological evaluation of the kidney was performed. Results: Kidneys of SHR did not develop signs of relevant histopathological changes, but showed increased glomerular area and cellularity. Plasma creatinine was decreased, and creatinine clearance was augmented in SHR at the end of the study. Urinary excretion of Klotho was higher in SHR at 5 and 8 months old, whereas plasma Klotho levels were similar to WKY. GluAp, AlaAp, and DPP4 urinary activities were increased in SHR throughout the time-course study. A positive correlation between glomerular area and cellularity with creatinine clearance was observed. Urinary GluAp, AlaAp, DPP4, and Klotho showed positive correlations with SBP. Conclusions: GluAp, AlaAp, DPP4, and Klotho in the urine are useful tools for the evaluation of renal damage at early stages, before the whole histopathological and biochemical manifestations of renal disease are established. Moreover, these observations may represent a novel and noninvasive diagnostic approach to assess the evolution of kidney function in hypertension and other chronic diseases.

To be, or not to be, that is the question. (William Shakespeare, Hamlet) Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2, respectively) play a role in trimming peptides that are too long to be bound and presented by class I HLA (HLA-I) molecules to CD8+ T cells. They may also affect the HLA-I-presented peptide repertoire by overtrimming potential epitopes. Both enzymes may also be released from the cell to cleave cytokine receptors and regulate blood pressure. Both enzymes are polymorphic, which affects their expression, specificity, and activity, resulting in their role in diseases associated with HLA-I. In this brief review, we concentrate on ERAP2, less investigated because of its lack in laboratory mice and 25% of humans, as well as a lower polymorphism. ERAP2 was found to be associated with several diseases and to influence ERAP1 effects. It was discovered recently that the defective ERAP2 gene, not encoding functional aminopeptidase, may nevertheless, during viral infections, produce a truncated protein isoform of unknown function, possibly interfering with ERAP1 and full-length ERAP2 by heterodimer formation. The disease associations of ERAP2, alone or in combination with ERAP1, are reviewed. [ABSTRACT FROM AUTHOR]

We performed a genome-wide association study of 6447 bipolar disorder (BD) cases and 12 639 controls from the International Cohort Collection for Bipolar Disorder (ICCBD). Meta-analysis was performed with prior results from the Psychiatric Genomics Consortium Bipolar Disorder Working Group for a combined sample of 13 902 cases and 19 279 controls. We identified eight genome-wide significant, associated regions, including a novel associated region on chromosome 10 (rs10884920; P=3.28 × 10-8) that includes the brain-enriched cytoskeleton protein adducin 3 (ADD3), a non-coding RNA, and a neuropeptide-specific aminopeptidase P (XPNPEP1). Our large sample size allowed us to test the heritability and genetic correlation of BD subtypes and investigate their genetic overlap with schizophrenia and major depressive disorder. We found a significant difference in heritability of the two most common forms of BD (BD I SNP-h2=0.35; BD II SNP-h2=0.25; P=0.02). The genetic correlation between BD I and BD II was 0.78, whereas the genetic correlation was 0.97 when BD cohorts containing both types were compared. In addition, we demonstrated a significantly greater load of polygenic risk alleles for schizophrenia and BD in patients with BD I compared with patients with BD II, and a greater load of schizophrenia risk alleles in patients with the bipolar type of schizoaffective disorder compared with patients with either BD I or BD II. These results point to a partial difference in the genetic architecture of BD subtypes as currently defined.

Telomeres are specialized nucleoprotein complexes that comprise the ends of linear chromosomes. Human telomeres end in a short, single-stranded DNA (ssDNA) overhang that is recognized and bound by two telomere proteins, POT1 and TPP1. Whereas POT1 binds directly to telomere ssDNA, its interaction with TPP1 is essential for localization of POT1 to the telomere. TPP1 also provides enhanced binding and sequence discrimination that regulates POT1-TPP1 interactions exclusively with telomere ssDNA. Finally, TPP1 recruits telomerase, the enzyme responsible for synthesis of telomere DNA, to the telomere. While the oligosaccharide-oligonucleotide-binding (OB)-fold domain of TPP1 has been solved by X-ray crystallography, the molecular interactions within the POT1-TPP1-ssDNA ternary complex and the conformational changes that contribute to its diverse functions remain ambiguous. We employed hydrogen/deuterium exchange combined with mass spectrometry to identify three peptides, all residing within the OB-fold of TPP1, that exhibit altered exchange rates upon complex formation or ssDNA binding. Mutation of these regions combined with functional assays revealed the diverse contributions of each moiety in protein-protein interactions, regulating telomerase activity or DNA-binding. Together, these functional data combined with biophysical analyses and homology modeling provide a molecular understanding of the diverse contributions of TPP1 in telomere maintenance.

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions. [ABSTRACT FROM AUTHOR]

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 μM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. Key points: • A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis– and C. glutamicum–based expression systems are built to promote secretory (soluble) Amp0279 expression. [ABSTRACT FROM AUTHOR]

Abstract Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide‐conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal‐derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA‐MB231. The tumorigenic D492HER2 and MDA‐MB231 cells were more sensitive than normal‐derived D492 cells when treated with melflufen. Compared to the commonly used anti‐cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA‐MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti‐neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.

A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and Pep X) activities, were evaluated. Most of the strains demonstrated salt tolerance to 6% of NaCl, while only two L. delbruekii (P14, P38), one L. rhamnosus (P50) and one L. plantarum (Q3C4) were able to grow in the presence of 10% (w/v) of NaCl. Except for 2 L. plantarum (Q1C6 and Q3C4), all strains showed low or medium acidifying activity and good proteolytic features. Furthermore, lipolytic activity was revealed in none of the strains, while the production of EPS and diacetyl was widespread and variable among the tested strains. Finally, regarding aminopeptidase activities, 1 L. delbrueckii (P10), 1 L. rhamnosus (P50), and 1 L. lactis (Q5C6) were considered as the better performing, showing high values of both Pep N and Pep X. Based on data presented here, the aforementioned strains could be suggested as promising adjunct cultures in cheesemaking.

UnlabelledBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.ImportancePeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between shelterin connectivity and tumorigenicity.

Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.

Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,647 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype.

Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study, we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 = 105 nM). Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of immune responses.