Precursor Polypeptide Research Articles

Female fertility and the mammalian egg's zona pellucida.

All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked fibrils that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along fibrils. Fibrils are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP fibrils fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 result in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.

Genome-wide identification of phytosulfokine (PSK) peptide family reveals TaPSK1 gene involved in grain development of wheat (Triticum aestivum L.)

BackgroundPhytosulfokine (PSK) functions as a plant peptide growth factor that plays important and diverse roles in plant development and stress responses. Nevertheless, PSKs have not been systematically analyzed in wheat.ResultsA genome-wide comparative analysis of PSK genes in wheat was conducted and 15 TaPSKs were identified and divided into four subgroups in the wheat genome based on sequence similarity. The examination of motif compositions of TaPSK genes revealed the presence of the YIYTQ signature motif in the C-terminus of all TaPSK polypeptide precursors, with a highly conserved feature across different species. Exogenous application of TaPSK peptide promoted root growth in wheat. Quantitative RT-PCR analysis revealed that the TaPSKs exhibited preferential or tissue-specific expression patterns in wheat. In particular, three homologs of the TaPSK1 genes were specifically expressed in grains, with the strongest expression observed in the developing grains at 15 days after anthesis. Compared with wild type, transgenic rice lines overexpressing TaPSK1-A exhibited larger grain size and higher thousand-grain weight. The promoter region and genomic sequence of the wheat TaPSK1-A gene were cloned. Sequence polymorphism showed that five single-nucleotide polymorphisms (SNPs) were identified in the promoter region of TaPSK1-A. A Kompetitive Allele-Specific PCR (KASP) marker was developed for TaPSK1-A based on –806 bp SNP (C/T transition), and two haplotypes, TaPSK1-A-HapI and TaPSK1-A-HapII were detected in 260 wheat accessions collected from different regions. The expression of TaPSK1-A, promoter activity, and thousand-grain weight (TGW) in the TaPSK1-A-HapII haplotype were higher than those in the TaPSK1-A-HapI haplotype. Furthermore, yeast one-hybrid assays revealed the binding of TaNF-YB1 and TaERF39 to the promoter regions of the TaPSK1-A gene, and TaMADS29 could bind to the promoters of TaPSK1-B and TaPSK1-D genes.ConclusionsComparative genome-wide analysis of TaPSK peptide family revealed that the TaPSK1 gene is involved in wheat grain development, and the developed TaPSK1-A-KASP marker could be utilized for marker-assisted selection breeding of wheat.Graphical

Ligation of multiple protein domains using orthogonal inteins with non-native splice junctions.

Protein splicing is a self-catalyzed process in which an internal protein domain (the intein) is excised from its flanking sequences, linking them together with a canonical peptide bond. Trans-inteins are separated in two different precursor polypeptide chains that must assemble to catalytically self-excise and ligate the corresponding flanking exteins to join even when expressed separately either in vitro or in vivo. They are very interesting to construct full proteins from separate domains because their common small size favors chemical synthesis approaches. Therefore, trans-inteins have multiple applications such as protein modification and purification, structural characterization of protein domains or production of intein-based biosensors, among others. For many of these applications, when using more than one trans-intein, orthogonality between them is a critical issue to ensure the proper ligation of the exteins. Here, we confirm the orthogonality (lack of cross-reactivity) of four different trans- or split inteins, gp41-1, gp41-8, IMPDH-1 and NrdJ-1 both in vivo and in vitro, and built different constructs that allow for the sequential fusion of up to four protein fragments into one final spliced product. We have characterized the splicing efficiency of these constructs. All harbor non-native extein residues at the splice junction between the trans-intein and the neighboring exteins, except for the essential Ser + 1. Our results show that it is possible to ligate four different protein domains using inteins gp41-1, IMPDH-1 and NrdJ-1 with non-native extein residues to obtain a final four-domain spliced product with a not negligible yield that keeps its native sequence.

Enzyme-responsive oncolytic polypeptide for tumor therapy

Host defense peptide-mimicking cationic oncolytic polymers have attracted increasing attention for cancer treatment in recent years. However, polymers with large amounts of positive charge may cause rapid clearance and severe off-target toxicity. To facilitate in vivo application, an alkaline phosphatase (ALP)-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been reported in this work. C12-PLL/PA could be hydrolyzed into the active form of the oncolytic polypeptide (C12-PLL) by the extracellular alkaline phosphatase within solid tumors, thereby resulting in the conversion of the negative charge to positive charge and restoring its membrane-lytic activity. Detailed mechanistic studies showed that C12-PLL/PA could effectively destroy cancer cell membranes and subsequently result in rapid necrosis of cancer cells. More importantly, C12-PLL/PA significantly inhibited the tumor growth in the 4T1 orthotopic breast tumor model with negligible side effects. In summary, these findings demonstrated that the shielding of the amino groups with phosphate groups represents a secure and effective strategy to develop cationic oncolytic polypeptide, which represents a valuable reference for the design of enzyme-activated oncolytic polymers. Statement of significanceRecently, there has been a growing interest in fabricating host defense peptide-mimicking cationic oncolytic polymers for cancer therapy. However, there remain concerns about the tumor selectivity and off-target toxicity of these cationic polymers. In this study, an alkaline phosphatase-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been developed to selectively target cancer cells while sparing normal cells. Mechanistic investigations demonstrated that C12-PLL/PA effectively disrupted cancer cell membranes, leading to rapid necrosis. Both in vitro and in vivo experiments showed promising anticancer activity and reliable safety of C12-PLL/PA. The findings suggest that this synthetic enzyme-responsive polypeptide holds potential as a tumor-specific oncolytic polymer, paving the way for future applications in cancer therapy.

Gonadotropin-inhibitory hormone and its receptors in teleosts: Physiological roles and mechanisms of actions

Gonadotropin-inhibitory hormone (GnIH) was the first reported hypothalamic neuropeptide inhibiting reproduction in vertebrates. Since its discovery in the quail brain, its orthologs have been identified in a variety of vertebrate species and even protochordates. Depending on the species, the GnIH precursor polypeptides comprise two, three or four mature peptides of the RFamide family. It has been well documented that GnIH inhibits reproduction at the brain-pituitary–gonadal levels and participates in metabolism, stress response, and social behaviors in birds and mammals. However, most studies in fish have mainly been focused on the physiological roles of GnIH in the control of reproduction and results obtained are in some cases conflicting, leaving aside its potential roles in the regulation of other functions. In this manuscript we summarize the information available in fish with respect to the structural diversity of GnIH peptides and functional roles of GnIH in reproduction and other physiological processes. We also highlight the molecular mechanisms of GnIH actions on target cells and possible interactions with other neuroendocrine factors.

Screening of Diabetes-Associated Autoantigens and Serum Antibody Profiles Using a Phage Display System.

Aims/Introduction: Phage display method is a crucial tool to find novel clinically valuable diabetes-associated autoantigens and identify known autoantigen epitopes that are associated with diabetes and could provide scientific support and guidance for the artificial construction and synthesis of Type I diabetes mellitus (T1DM) novel biomarkers. Materials and Methods: The phage display system was used for the "biopanning" of T1DM serum. Following the sequencing of the phage DNAs, the homologous sequences of the above fusion heptapeptide were further investigated by BLAST to track the origin of the polypeptide sequences. The antibody spectrum revealed new T1DM-associated epitopes and antibodies. Results: A total of 1200 phage DNA were sequenced and 9 conserved polypeptide sequences were collected. It was confirmed that the zinc transporter and islet amyloid protease were among them. The conserved polypeptide sequence 8 and another three distinctive polypeptide sequences derived from Proteus were discovered. Furthermore, we expressed recombinant proteins with homologous polypeptide sequences for the human islet amyloid polypeptide (IAPP) and polypeptide precursor human zinc transporter 8 (ZNT8). Through clinical sample detection for the serum from T1DM (n = 100) and T2DM (n = 200) patients, results demonstrate the importance and relevance of these polypeptides in the recognition and classification of various forms of diabetes. Conclusion: Human pancreatic and concurrent bacterial-derived protein antigens and their epitopes were identified in this research by the phage display system, which is crucial for distinguishing different types of diabetes.

SAT268 Regulation Of Stem/Progenitor Cells Of The HPA Axis During Stress Adaptation

Abstract Disclosure: P. Siatra: None. D. Cozma: None. I. Berger: None. I. Oikonomakos: None. M. Theodoropoulou: None. S.R. Bornstein: None. C.L. Andoniadou: None. C. Steenblock: None. Chronic stress is a pervasive concern in the modern society. A long-term hyper activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to elevated amounts of stress hormones [e.g. ACTH and glucocorticoids (GCs)]. This can incur maladaptation that eventually contributes to mental illness, cardiovascular dysfunctions, diabetes, cancer and autoimmune diseases. Emerging key players for stress adaptation of the HPA axis are stem/progenitor cell populations. In this study, we aim to unravel the mechanisms involved in the regulation of stem/progenitor cells of the HPA axis during stress adaptation. To that aim, we incorporated an in vitro system of non-adherent spheroids from primary adrenocortical mouse cells that were stimulated with ACTH. The treatment resulted in the increased growth of the spheroids compared to unstimulated controls indicating a potential role of the hormone in cell proliferation and stem cell behaviour. This result is in line with the enlarged adrenals observed in patients with Cushing’s syndrome. To assess the contribution of stem cells during stress adaptation in the adrenal cortex in vivo, we performed immobilization stress and lineage tracing studies using the tamoxifen inducible Gli1:CreERT2/R26R:eYFP mouse line. By combining these results along with single-cell RNA-seq technology, we aim to shed light into molecular factors involved in the adult adrenal cortex regeneration and remodelling. To follow the effect of GC excesses on the negative feedback loop observed during a normal stress response on the pituitary gland, we stimulated adherent primary anterior pituitary colonies with dexamethasone and corticosterone. GC treatment led to downregulation of Pomc expression, a precursor polypeptide for ACTH and other hormones, in the treated colonies compared to the control. This downregulation caused by GCs could be mediated through different gene targets of the glucocorticoid receptor as well as via paracrine signalling from pituitary stem cells. Altogether, our results will provide us with more profound knowledge of mechanisms underlying interactions between stressors and stem/progenitor cells of the HPA axis along with novel therapeutic targets for stress-associated disorders. Presentation: Saturday, June 17, 2023

Molecular cloning and bioinformatic characterization of Gonadotropin Inhibitory Hormone (GnIH) and its receptors in the freshwater murrel, Channa punctatus (Bloch, 1793).

Gonadotropin inhibitory hormone belonging to the RFamide peptide family, a hypothalamic neuropeptide, regulates Hypothalamus-pituitary-gonadal (HPG) axis and inhibits gonadal development. GnIH polypeptide precursor has an Arg-Phe-NH2 (RFamide) motif at the C-terminal, which has LPXRF (X = Q or L) domain. The actions of GnIH are mediated through G-protein coupled receptors and upto threereceptors have been characterized in many teleosts. GnIH exerts its inhibitory effect on the HPG axis through direct interaction with GnRH and Kisspeptin neurons in the brain and acts directly on the pituitary gonadotrophs. To decipher the role of GnIH in Indian freshwater murrel, Channa punctatus, we sequenced the cDNA encoding GnIH and its two receptors. The identified GnIH mRNA encodes three RFamide peptides having -MPMRF, -MPQRF, and -LPQRFamide motifs. In silico analysis of the amino acid sequence of GnIH exhibits its molecular and functional properties and the protein-protein interaction with significant factors regulating the HPG axis. The 3-D structure of GnIH and its receptors, provides more relevant information about the active residues of these proteins which might be involved in their functioning and interaction with other proteins. Molecular dynamic simulation of GnIH protein has provided more insight into its dynamic behavior. The expression of GnIH and its receptors, shows an inverse correlation with gonadal development during the annual reproductive cycle.

RPS27a and RPL40, Which Are Produced as Ubiquitin Fusion Proteins, Are Not Essential for p53 Signalling.

Two of the four human ubiquitin-encoding genes express ubiquitin as an N-terminal fusion precursor polypeptide, with either ribosomal protein (RP) RPS27a or RPL40 at the C-terminus. RPS27a and RPL40 have been proposed to be important for the induction of the tumour suppressor p53 in response to defects in ribosome biogenesis, suggesting that they may play a role in the coordination of ribosome production, ubiquitin levels and p53 signalling. Here, we report that RPS27a is cleaved from the ubiquitin-RP precursor in a process that appears independent of ribosome biogenesis. In contrast to other RPs, the knockdown of either RPS27a or RPL40 did not stabilise the tumour suppressor p53 in U2OS cells. Knockdown of neither protein blocked p53 stabilisation following inhibition of ribosome biogenesis by actinomycin D, indicating that they are not needed for p53 signalling in these cells. However, the knockdown of both RPS27a and RPL40 in MCF7 and LNCaP cells robustly induced p53, consistent with observations made with the majority of other RPs. Importantly, RPS27a and RPL40 are needed for rRNA production in all cell lines tested. Our data suggest that the role of RPS27a and RPL40 in p53 signalling, but not their importance in ribosome biogenesis, differs between cell types.

Characterisation of capsid polypeptide P1 and capsid protein VP1 of the Malaysia foot and mouth disease virus (FMDV) serotype O and A isolates

Foot and mouth disease virus (FMDV) is the cause of foot and mouth disease (FMD) outbreaks in livestock worldwide, which affects domestic and international trade, resulting in significant economic losses and social consequences. For efficient monitoring and prevention of FMD outbreaks, the need for improved strategies to control FMDV and achieve FMD-free status with various control measures including vaccination can be established. In vaccinology, major advances and discoveries in vaccination variations including DNA and protein subunit vaccines proved to be more economical and sustainable. To develop a safe vaccine for animals, possible antigenic genes or antigens need to be identified and characterised. The FMDV is a single-stranded RNA virus consisting of a capsid precursor polypeptide, P1, which encodes for four structural proteins (VP4-1), leading to antigenic variation and VP1 potentially carrying the key epitope for vaccine development. This study aims to identify and characterise the capsid polypeptide, P1 and capsid protein, VP1 of the Malaysian FMDV serotype O and serotype A isolates. The nucleotide and protein sequences were identified based on the FMD outbreaks in Malaysia and the antigenicity of the P1 and VP1 was predicted by Kolaskar and Tongaonkar's semi-empirical method. Subsequently, the P1 and VP1 genes were inserted into pET-28a, respectively, and used for protein expression analysis. The P1 and VP1 were predicted to be antigenic via in silico analysis and successfully expressed and characterised through in vitro analysis. Hence, this study can be exploited as a tool to design a new novel vaccine for vaccine development against FMD in bovines.

An artificially split class 3 intein.

Inteins excise themselves from precursor polypeptides through protein splicing, joining N- and C-exteins with a peptide bond. Split inteins are expressed as separate polypeptides that undergo protein trans splicing (PTS). Here, we demonstrate PTS can be achieved using an artificially split class 3 intein. Because class 3 inteins use an internal initiating nucleophile near the C-extein junction, rather than the first residue of the intein, both catalytic nucleophiles are present on a single polypeptide. This results in a compact arrangement of catalytic nucleophiles for PTS compared to the standard arrangement for split class 1 inteins.

Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55Gag

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

Values of PCT in neonates.

Procalciton (PCT) is a precursor polypeptide of the hormone calcitonin, produced in C cells of the thyroid. It has been demonstrated that microbial toxins and proinflammatory mediators can cause the release of PCT from tissues and cells in the body. PCT thus has become an important marker in the diagnosis of infection. In this retrospective study we analyzed blood samples performed for clinical purposes from the newborns present in our hospital in the year 2019. We developed a database of 1356 PCT values obtained from 224 infants at risk for neonatal infection; we selected those PCT values obtained within 24 hours from a blood sampling for blood culture. Babies with positive blood culture had PCT values more elevated than those with negative blood culture (17.061 ng/mL [C.I. 10.8-23.2] vs. 4.6 ng/mL [C.I. 2.6-6.6]). No statistically significant difference was found between babies with negative blood culture born before or after 37 weeks of gestation. This paper gives useful data of PCT values in non-infective babies. It is worth to show that the normality values should not be confused with those of older children or adults. Moreover, it shows the reliability of PCT as an infection index.

Structure of AQEE-30 of VGF Neuropeptide in Membrane-Mimicking Environments.

AQEE-30 is one of the VGF peptides, which are derived from the VGF polypeptide precursor, and related to various physiological phenomena including neuroprotective effects in Huntington's disease and amyotrophic lateral sclerosis (ALS). Although various functions of AQEE-30 have been reported so far, the structure of this peptide has not been reported yet. In this study, the structure of human AQEE-30 was investigated in hexafluoroisopropanol (HFIP) and dodecyl phosphocholine (DPC) micelle solutions, using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AQEE-30 had a partial helical structure in aqueous buffer, and the helical structure was stabilized in the HFIP and DPC micelle solutions. The 3D structures determined by NMR spectroscopy showed that AQEE-30 adopted mainly α-helical structure in both the HFIP and DPC micelle solutions. The surface of AQEE-30 showed that it was predominantly negatively charged. The residues from 601 to 611 in both the HFIP and DPC micelle solutions showed amphiphilicity with four negatively charged residues, glutamate. The C-terminal consecutive arginine residues formed a partial positively charged surface. These results suggest an α-helical active structure of AQEE-30 in the cell-membrane environment.

Organic solvents aggregating and shaping structural folding of protein, a case study of the protease enzyme

Low solubility of reactants or products in aqueous solutions can result in the enzymatic catalytic reactions that can occur in non-aqueous solutions. In current study we investigated aqueous solutions containing different organic solvents / deep eutectic solvents (DESs) that can influence the protease enzyme's activity, structural, and thermal stabilities. Retroviral aspartic protease enzyme is responsible for the cleavage of the polypeptide precursors into mature viral components, a very crucial step for virus life cycle. In molecular dynamic simulations (MDS), the complex of the protease enzyme with Darunavirwas found highly stable in urea aqueous solution compared to when with the ethylene glycol (EG) or glycerol solvents. Particularly, in different organic solvents the presence of Darunavir induced protein-protein interactions within the protease homodimer. For the systems with EG or glycerol solvents, the flap domains of the enzyme formed an “open” conformation which lead to a weak binding affinity with the drug. Conserved D25 and G27 residues among this family of the aspartic protease enzymes made a stable binding with Darunavir in the urea systems. Unfolding of the protease dimer was initiated due to self-aggregation for the EG or glycerol organic solvents, which formed an “open” conformation for the flap domains. On the contrary lack of such clustering in urea solvent, the protease showed conventional structural folding in the presence or absence of the drug molecule. These novel findings may help to better understand the protease enzymes, which could be controlled by deep eutectic solvents.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting.

Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together invitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import.

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Inverse Correlation of KISS1 and KISS1R Expression in Triple-negative Breast Carcinomas from African American Women.

The kisspeptin 1 (KISS1) gene encodes a precursor polypeptide which after proteolysis forms the kisspeptin-10 (KISS1) protein. KISS1, retains maximum physiological activity when it binds to its receptor (KISS1R), allowing KISS1 to effectively function as a suppressor of metastasis in melanomas and other types of cancer. The goal of this study was to evaluate the expression of KISS1 and KISS1R in breast carcinomas from African American (AA) women and correlate their association with clinicopathological features, including breast cancer subtypes, and outcomes. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded surgical blocks from 216 AA patients. KISS1 and KISS1R expression was assessed using immunohistochemistry. Univariate analysis was used to determine the association between the expression of KISS1 and KISS1R, and clinicopathological characteristics. Pearson correlation was also determined between immunohistochemical H-scores, tumor size, and the number of positive lymph nodes. Kaplan-Meier estimates of overall and disease-free survival were plotted, and log-rank tests were performed to compare estimates among groups. KISS1 protein expression was found to be higher in receptor-negative and triple-negative breast cancer (TNBC) compared to other subtypes (p<0.001). However, KISS1R expression was higher in non-TNBC tumors compared to other subtypes (p<0.001). Higher KISS1R expression was marginally negatively correlated with tumor size (p=0.077), and positively correlated with lymph-node positivity (p=0.056), and disease-free survival (p=0.092). Our study showed a significant inverse correlation between KISS1 and KISS1R in TNBC. This investigation implicates a role for KISS1 and KISS1R in the pathogenesis of TNBCs in AA women.

The genetic properties accompanied of "quasi-species" crossing of Small Ruminant LentiViruses (SRLVs)

The env gene of Small Ruminant LentiViruses (SRLV) encodes a polypeptide precursor which after glycosylation is cleaved to give the two glycoproteins of the viral envelope: the transmembrane protein (TM) and the surface protein (SU). The passage and adaptation of SRLVs in new hosts is always accompanied by genetic mutations in the SU region of the env gene that determines the tropism of the virus by recognition of the cellular receptor. We studied the genetic properties of SRLVs accompanied of the "quasi-species" in Capra ibex. 16 Blood samples of Capra ibex were tested for presence of specific antibodies against SRLVs using a commercially available ELISA. Peripheral blood mononuclear cells (PBMC) isolated on Ficoll gradient were cultured in macrophage differentiation medium to obtain monocyte-derived macrophage (MDM) monolayers for virus isolation. DNAs from non cultured PBMC were used as templates for PCR and Nested PCR amplification of proviral genome. Nested PCR products (108 nt) were cloned and sequenced. Sequences were analysed using ClustalW software. The alignments of the SU region sequences show three important types of genetic mutations: deletion, addition and replacement nucleotides. We present data showing that the sequences of C. ibex SRLV isolates are closer to the prototypic CAEV-Co isolate. This might indicate a recent "quasi-species" infection of SRLV in C. ibex.