Podcasts about peptidase

Commentary by Dr Chung-Lieh Hung

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.28.518246v1?rss=1 Authors: Dulloo, I., Tellier, M., Levet, C., Chikh, A., Zhang, B., Webb, C. M., Kelsell, D. P., Freeman, M. Abstract: iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signalling; they function primarily by recognising transmembrane domains of their clients. Here we report an unexpected, and mechanistically distinct, nuclear function of iRhoms. iRhom2 is a non-canonical substrate of the signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the cellular transcriptome. We observed elevated nuclear iRhom2 in skin biopsies of patients with psoriasis and tylosis with oesophageal cancer (TOC); increased SPC-mediated iRhom2 cleavage in a psoriasis model; and overlapping transcriptional signatures between psoriasis and expression of the iRhom2 N-terminus. This work highlights the pathophysiological significance of this SPC-dependent ER-to-nucleus signalling pathway, and is the first example of a rhomboid-like protein that mediates protease-regulated nuclear signalling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513473v1?rss=1 Authors: Maccioni, R., Travisan, C., Zerial, S., Wagener, A., Andrade-Talavera, Y., Picciau, F., Grassi, C., Chen, G., Lemoine, L., Fisahn, A., Jiang, R., Fluhrer, R., Mentrup, T., Schröder, B., Nilsson, P., Tambaro, S. Abstract: Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid {beta}-peptide (A{beta}) levels. To identify new druggable pathways involved in the A{beta} cascade we here investigated the AD pathophysiological role of the presenilin-like intramembrane protease signal peptide peptidase-like 2b (SPPL2b). A{beta}42 induced a biphasic modulation of SPPL2b expression in human cell lines and ex vivo mouse brain slices. In addition, SPPL2b was elevated in AppNL-G-F knock-in AD mice as well as in human AD samples. Early high neuronal expression of SPPL2b was followed by a downregulation in late AD pathology in both AppNL-G-F mice and Braak stage V AD brains. Importantly, SPPL2b overexpression or its genetic deletion significantly increased or reduced APP cleavage and A{beta} production, respectively. Thus, our results strongly support the involvement of SPPL2b in AD pathology. The early A{beta}-induced SPPL2b upregulation may enhance A{beta} production in a vicious cycle further aggravating the A{beta} pathology suggesting SPPL2b as a potential anti-A{beta} drug target. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

What an amazing discovery these drugs are. With novel ways of beating diabetes, they go beyond just insulin secretion, FASCINATING! Listen up to how our bodies work then apply these drugs.

Commentary by Dr. Douglas Mann

As individuals, we are all exposed to a variety of substances on a daily basis, from the foods we eat to the household and hygienic products we use to environmental agents. This constant exposure can cause the bloodstream to become overloaded with allergens, inflammatory agents (e.g., bacteria, fungi, antigens), undigested food, and even fibrin, which is a blood clot-forming protein. The buildup of these types of substances can put a strain on the immune system and subsequently lead to the increased production of pro-inflammatory proteins that can worsen pain or discomfort, making it harder to recover and cause immune hypersensitivity. Certain remedies may cause side effects and even weaken the immune response by design (such as allergy and arthritis medications), while others can target harmful invaders, promote cleansing and detoxification, and strengthen the immune system.One beneficial range of natural remedies that can support a healthy immune and inflammatory response are proteolytic enzymes. Beneficial proteolytic enzymes include peptidase, nattokinase, protease, papain, and bromelain. These enzymes support the complete digestion of proteins from foods, and certain other circulating protein particles. They may even help cleanse the body of harmful toxins and microorganisms.When there is an excessive buildup of undigested food (e.g., sugars, fats, proteins, etc.), toxins, fibrin, and infectious agents, these substances can be transferred to the large intestines or bloodstream where antibodies attach to them and create what are known as circulating immune complexes (CICs). Antibodies bind to undigested food particles and other harmful substances as a means of signaling the immune system to increase the production of white blood cells that can seek out and destroy them. The human body needs food (macronutrients) for energy, growth and repair and to keep warm. We need many nutrients on a daily basis in order to stay healthy. The three main nutrient groups in food are carbohydrates, protein and fats. A normal digestive system can quickly convert macronutrients into micronutrients like amino acids, vitamins, and minerals. A less than optimal digestive system may not properly convert foods into nutrients. This may lead to a condition known as leaky gut (LG). With LG, partially digested foods may enter the circulatory system and begin to decompose into CIC’s. High levels of CICs can put a strain on the immune system and reduce its ability to fight off other infectious agents. Accordingly, CICs are linked to the onset of various complications [8]. More specifically, the accumulation of CICs may cause abnormal cortisol levels as well as chronic systemic inflammation that is associated with the onset of cardiovascular, blood sugar, memory, and even metabolic issues.Peptidase and nattokinase, in particular, target substances that cause fluid accumulation, inflammation, and swelling. This leads to rapid drainage and cleansing away from the affected region. This process also shortens the recovery period. Peptidase is especially unique as this enzyme is produced by non-pathogenic bacteria called Serratia sp. E-15, which is localized in the intestines of silkworms. This powerful enzyme breaks down the walls of the chrysalis that silkworms grow in as they begin to undergo metamorphosis, the process through which they become moths. Based on this discovery, it was proposed that this enzyme is capable of breaking down dead tissue without damaging an organism’s healthy cells. This property heightens immunity by promoting the destruction and detoxification of harmful substances that the immune system is normally tasked with addressing.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378711v1?rss=1 Authors: Liaci, A. M., Steigenberger, B., Tamara, S., Telles de Souza, P. C., Grollers-Mulderij, M., Ogrissek, P., Marrink, S.-J., Scheltema, R. A., Foerster, F. Abstract: The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. This unique architecture generates specificity for thousands of SPs based on the length of their hydrophobic segments. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.24.265033v1?rss=1 Authors: Silva-Garcia, M., Bolgi, O., Ross, B., Pilla, E., Vijayalakshmi, K., Killisch, M., Stark, N., Lenz, C., Spitzner, M., Gorrell, M. D., Grade, M., Urlaub, H., Dobbelstein, M., Huber, R., Geiss-Friedlander, R. Abstract: Dipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development. Copy rights belong to original authors. Visit the link for more info

Betrachtungen und Reflektionen zu Peptidase. Erfahre einiges zum Thema Peptidase in diesem Audio Podcast. Eine Ausgabe des Naturheilkunde Podcasts von und mit Sukadev Bretz, Yogalehrer bei Yoga Vidya. Anmerkung: Gesundheitliche Informationen in diesem Podcast sind nicht gedacht für Selbstdiagnose und Selbstbehandlung, sondern Gedankenanstöße aus dem Gebiet der Naturheilkunde. Bei eigener Erkrankung brauchst du Arzt oder … „Peptidase“ weiterlesen

Ein Podcast zu was, wozu und warum Peptidase. Erfahre einiges über Peptidase in dieser Vorlesung. Diese Podcastsendung ist eine Ausgabe des Naturheilkunde Podcast. Sie ist erstellt worden als Basis für einen Lexikonartikel im Yoga Wiki Naturheilkunde Lexikon von A-Z von Yoga Vidya. Der Yogalehrer Sukadev interpretiert hier das Wort bzw. den Ausdruck Peptidase aus dem Geist des ganzheitlichen Yoga, also aus einer Einstellung der Achtsamkeit. Peptidase kommt aus Themengebieten wie Heilsteine, Mineral. Nicht alles sollte man zu ernst nehmen. Lachen ist die beste Medizin. Wie du diese anwenden kannst, erfährst du in der Lachyoga Übungsleiter Ausbildung. Welche Meinung kommen dir dazu? Wir freuen uns über Ergänzungen in den Kommentaren. Anmerkung: Gesundheitliche Informationen in diesem Podcast sind nicht gedacht für Selbstdiagnose und Selbstbehandlung, sondern Gedankenanstöße. Bei eigener Erkrankung brauchst du Arzt oder Heilpraktiker.

Ein Podcast zu was, wozu und warum Peptidase. Erfahre einiges über Peptidase in dieser Vorlesung. Diese Podcastsendung ist eine Ausgabe des Naturheilkunde Podcast. Sie ist erstellt worden als Basis für einen Lexikonartikel im Yoga Wiki Naturheilkunde Lexikon von A-Z von Yoga Vidya. Der Yogalehrer Sukadev interpretiert hier das Wort bzw. den Ausdruck Peptidase aus dem Geist des ganzheitlichen Yoga, also aus einer Einstellung der Achtsamkeit. Peptidase kommt aus Themengebieten wie Heilsteine, Mineral. Nicht alles sollte man zu ernst nehmen. Lachen ist die beste Medizin. Wie du diese anwenden kannst, erfährst du in der Lachyoga Übungsleiter Ausbildung. Welche Meinung kommen dir dazu? Wir freuen uns über Ergänzungen in den Kommentaren. Anmerkung: Gesundheitliche Informationen in diesem Podcast sind nicht gedacht für Selbstdiagnose und Selbstbehandlung, sondern Gedankenanstöße. Bei eigener Erkrankung brauchst du Arzt oder Heilpraktiker.

Digestive enzymes might be the most overlooked, underappreciated part of a good nutrition plan. Even the healthiest diet does little if you don't have good digestive health.  Unfortunately, most people don't think about using them unless they have issues with their gut health. As you'll see, they're essential for more than breaking down the food you eat. They can play a role in weight loss, muscle growth, and aging well. Not surprisingly, they're part of my Foundational Five. About 20% of the US population is known to have a digestive issue. That's about 60-70 million people. The percentage continues to grow in the US and across the world.  Symptoms can be as subtle as gas and bloating to more extreme symptoms like constant diarrhea or intermittent pain. Exercise, increased core body temperature, injury, stress, and certain diseases can compromise enzyme production. Processed foods may deplete enzyme activity and availability.  Digestive enzyme insufficiency may contribute to: Irritable Bowel Syndrome (IBS)hyperthyroidismCeliac diseaseCrohn's disease Excessive exercise and diets high in processed foods contribute to a lack of enzymes. What are Digestive Enzymes? Enzymes speed up chemical reactions in the body. You use more than 5000 different enzymes every day. Most of those enzymes are metabolic enzymes, responsible for everything from your thoughts to the thickness of your blood. A relatively small group of your 5000 enzymes convert the food you eat to nutrients that fuel and build your body. Without digestive enzymes, you wouldn't break down protein, fat, and carbohydrates, and your foods would pass through you undigested. Along the way, the food would destroy your intestines' lining, cause immune reactions, and cause inflammation. Nutritionally, you'd starve, no matter how much food you ate. Digestive enzymes fall into three different categories, based on the macronutrient they act on: Proteases and peptidases: Convert protein to peptides and amino acids. They also act on other parts of the body to support normal immune function, inflammation levels, tissue repair, and blood viscosity.Common proteases and peptidases: Bromelain, Pancreatin, Papain, Peptidase, Protease, TrypsinCarbohydrases: Convert carbohydrates to glucose and fructose.Common carbohydrases: Alpha-galactosidase, Amylase, Cellulase, Diastase, Glucoamylase, Invertase, Lactase, PhytaseLipases: Convert fat to fatty acids.Common lipases: Lingual Lipase, Gastric Lipase, Pancrealipase Proteases and Peptidases (Proteolytic Enzymes) Proteases (also known as proteolytic enzymes) act on protein in the digestive system. However, they also affect many other areas of the body. The average healthy adult breaks down 250-300 grams of protein throughout the body every day. Your body does this to replace damaged or aged tissues with new ones. Proteolytic enzymes play an essential role in this process. They also help maintain healthy inflammation levels, modulate pain, and support normal immune function. Because the body can produce a limited number of proteolytic enzymes, demand can exceed supply. Following an injury or extreme physical stress, proteolytic enzymes can be directed to the tissue repair, leaving the digestive system without enough to complete digestion. This could be why athletes often deal with digestive issues. If they don't get extra proteolytic enzymes through food or supplements, their available enzymes take part in tissue repair, leaving them short on what they need for proper digestion. On the other hand, in some people, enzymes are directed to digestion, leaving the rest of their body short. In this case, inflammation could get out of hand, or tissues and joints could get irritated. When supplemented in the diet, proteolytic enzymes have been shown to reduce stiffness and exercise-related soreness.* European practitioners regularly recommend proteolytic enzymes to support overall healt...

Hosts: Vincent Racaniello, Michele Swanson, and Michael Schmidt. Vincent, Michael, and Michele reveal how a fungal protease blunts the innate immune response and promotes pathogenicity. Subscribe to TWiM (free) on iTunes, Stitcher, RSS, or by email. You can also listen on your mobile device with the Microbeworld app. Links for this episode Michele on Flint Legionella outbreak (Detroit News) Fungal mimicry of a mammalian aminopeptidase (Cell Host Micr)   This episode is sponsored by ASM Agar Art Contest and ASM Microbe 2016 Send your microbiology questions and comments (email or mp3 file) to twim@twiv.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twim.

Intramembrane proteolysis - hydrolysis of membrane proteins within or close to their membrane-spanning regions - is a crucial cellular process that is conserved throughout all kingdoms of life. It is executed by distinct classes of polytopic membrane proteins, the intramembrane-cleaving proteases, that provide a hydrophilic, proteinaceous environment accommodating membrane protein substrates as well as water molecules within the hydrophobic membrane interior and catalyse peptide bond hydrolysis. In particular, intramembrane-cleaving aspartyl proteases have received attention as the presenilins, the catalytic subunits of the γ-secretase complex, were identified as key players in Alzheimer's disease pathophysiology. In addition to presenilins, mammalian genomes harbour presenilin homologues which include signal peptide peptidase (SPP) and SPP-like (SPPL) proteases. Among these, the Golgi-resident protease SPPL3 stands out as it is highly conserved among metazoa and SPPL3 orthologues are also found in plants. However, due to the lack of known substrates, SPPL3 has thus far hardly been characterised. Hence, the purpose of this study was to identify its substrates and elucidate its physiological function(s). In the first part of this study, the foamy virus envelope glycoprotein (FVenv) was identified as the first substrate of SPPL3. This allowed to study SPPL3's proteolytic activity in detail, with a focus on its substrate selectivity and sensitivity towards previously characterised inhibitors of intramembrane-cleaving aspartyl proteases. Importantly, this study revealed in addition that two other intramembrane-cleaving proteases, SPPL2a and SPPL2b, also endoproteolyse FVenv. SPPL2b in particular had been studied in detail before and therefore SPPL3- and SPPL2b- mediated endoproteolysis of FVenv were examined in parallel to directly compare these phylogenetically related intramembrane-cleaving proteases. This uncovered an unexpected idiosyncrasy of SPPL3 that clearly sets SPPL3 apart from other intramembrane-cleaving aspartyl proteases: SPPL3 endoproteolysed full-length FVenv and did not require the substrate's prior tailoring by another proteolytic activity - an otherwise common phenomenon among intramembrane-cleaving aspartyl proteases. In the second part, the physiological function of SPPL3 was investigated. Alterations in the cellular levels of proteolytically active SPPL3 turned out to impact the composition of N-glycans attached to endogenous cellular glycoproteins. SPPL3 over-expression was accompanied by a decrease in glycoprotein molecular weight, i.e. a hypoglycosylation phenotype, while loss of SPPL3 expression in cell culture models but also in vivo resulted in a hyperglycosylation phenotype. This led to the identification of Golgi glycan-modifying enzymes such as GnT-V and β3GnT1 as novel physiological substrates of SPPL3. Loss or reduction of SPPL3 expression, for instance, led to a marked intracellular accumulation of these enzymes, explaining the more extensive N-glycan elaboration and the hyperglycosylation phenotype observed under these conditions. At the same time secretion of these enzymes was reduced under these conditions. Together with additional observations such as the mapping of the SPPL3 cleavage site to the membrane-spanning region of GnT-V, this study demonstrates that SPPL3-mediated intramembrane proteolysis of such glycan-modifying enzymes liberates their active site-harbouring ectodomains. Acting in this manner, SPPL3 controls the intracellular pool of active glycan-modifying enzymes. Importantly, the finding that SPPL3 proteolytically cleaves full-length glycan-modifying enzymes and sheds their ectodomains is well in line with the observations made for FVenv and suggested that SPPL3 acts functionally equivalent to classical sheddases or rhomboid proteases but much unlike all other characterised mammalian intramembrane-cleaving aspartyl proteases. To examine whether these observations hold also true on a global cellular scale, a proteomic approach was undertaken in the third part of the study to define the SPPL3 degradome of HEK293 cells in conditions of SPPL3 over-expression. On the one hand, this led to the identification of numerous novel, mostly Golgi-resident candidate SPPL3 substrates and, considering the physiological implications, suggests that SPPL3 is very intricately linked to Golgi function. On the other hand, this approach supports the initial hypothesis that SPPL3 acts as a cellular type II membrane protein-selective sheddase. Taken together, this study provides the first in-depth characterisation of the intramembrane protease SPPL3 and reveals the cellular function of SPPL3. SPPL3 displays considerable and marked differences to other intramembrane-cleaving aspartyl proteases and emerges as a fundamental cellular sheddase that exhibits strong selectivity for type II-oriented, Golgi-resident membrane proteins. Products of SPPL3-mediated endoproteolysis of these Golgi factors are secreted and/or may be subject to intracellular degradation which compromises their catalytic activity. Thus, SPPL3 indirectly controls protein glycosylation in the Golgi apparatus.

Hosts: Vincent Racaniello, Alan Dove, and Kathy Spindler Vincent, Alan, Kathy, and Dickson discuss identification of a cell receptor for the coronavirus-EMC, and the role of interferon-epsilon in protecting the female reproductive tract. Links for this episode: Dipeptidyl peptidase 4 is CoV-EMC receptor (Nature) Receptor for new coronavirus (Nature) Broad reception for coronavirus (Nature) SARS 10th anniversary (Science) Interferon-epsilon protects female reproductive tract (Science) Families fighting flu (YouTube) Monitoring infections with online data (TWiM 41) Natalie Portman was a scientist (NY Times) Letters read on TWiV 223 Weekly Science Picks Kathy - 2013 Intel Science Talent Search awardsAlan - Krebs Cycle Rap (CDC)Vincent -  Jetpens (favorite one and two)D ickson - Rabies death in organ recipients (CDC) Listener Pick of the Week Justin - The DNA StoreJim - Massive Open Online ClassroomsChris - Feedly Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Peptidase Simple und komplexe Infos zum Thema Peptidase in diesem Kurzvortrag von Sukadev Bretz, dem Gründer von Yoga Vidya. Hier findest du: Seminare mit Sukadev Seminarübersicht Yoga Vidya YouTube Live Kanal Online Seminare Video Seminare Yoga Vidya kostenlose App Yoga Vidya Newsletter Kochrezepte Ayurvedische Ernährung Forum Onlineshop Schon ein kleiner Beitrag kann viel bewegen... Spende an Yoga Vidya e.V.! »

Orally and intravenously administered glucose yields comparable levels of glucose in plasma but different responses of insulin-release. Specialised mucosa cells react to glucose contents in chyme with release of glucagon-like peptide-1 (GLP-1) into the portal bloodstream, a peptide hormone, that makes beta-cells more sensitive to the subsequent glucose stimulus and thereby enhances the insulin-release exclusively under increased blood glucose levels without causing the insulin-release directly. The capacity of this entero-insular axis is limited primarily by the endogenous enzyme dipeptidyl peptidase IV (DPP-IV). Prevailingly the most progressive approach to therapy of non-insulin-dependent diabetes mellitus (NIDDM) in human medicine is targeted on the inhibition of DPP-IV, in order to raise GLP-1 concentration in plasma and to increase consequently the insulin-release in dependence on elevated blood glucose levels. In the limelight of discussion are the advantages of certain avoidance of insulin-caused hypoglycaemia on the one hand and the possibility of oral administration of the antienzyme on the other hand. In contrast to the condition in dogs, where the disease manifests almost exclusively in the form of secondary (insulin-dependent) diabetes mellitus, the type 2 (NIDDM) predominates in humans as well as in cats, whence it comes that the employment of oral antidiabetics in the latter species is quite promising, as the experience with the sulfonyl urea derivative glipizide has shown. As however the residual capacity of beta-cells of feline diabetics with regard to the release of endogenous insulin is subject to considerable variation, the risk of inducing hypoglycaemia is given by use of both insulin and sulfonyl urea derivatives, whereas this threat is circumvented by use of DPP-IV-inhibitors. In the present thesis the effect of DPP-IV inhibition on the elimination kinetics of GLP-1 is described by means of systematically combined infusion of the enzyme’s substrate (GLP-1) and its antienzyme in the rat model. The maximum effective dose of DPP-IV inhibitor is determined and the portion of hepatic clearance and whole blood clearance of total elimination of GLP-1 without concurrent DPP-IV inhibition is quantified approximately by means of isolated perfused rat livers and in-vitro-experiments. Finally the dependence of results on the respective experimental design is demonstrated. The total clearance of GLP-1 as substrate of DPP-IV without concurrent inhibition amounts to at least 57 ± 17 (mL/min)/kg and under influence of the DPP-IV-inhibitor to at least 22 ± 2 (mL/min)/kg. The value of clearance is highly dependent on the respective substrate concentration in plasma, so that the values range from 139 ± 57 (mL/min)/kg to 73 ± 15 (mL/min)/kg within the bounds of physiological GLP-1 concentrations in plasma. The maximum increase of GLP-1 levels in plasma (factor 3.4) on average of all GLP-1 infusion rates was attained by a DPP-IV inhibitor concentration in plasma of 0.4 µmol/L. The liver as central detoxication-organ figures out at 57 % (34/60) of total clearance, the soluble DPP-IV fraction in plasma however accounts for only about 1 % (0.7/60) of total clearance. The relatively low hepatic extraction ratio of 30 ± 11 % reversely allows seven out of ten GLP-1 molecules to enter the systemic circulation in spite of the first-pass-effect and thereby to reach possibly the receptors on the beta-cells (70 % bioavailability). The half-life of GLP-1 (7-36 amide) in the isolated perfused rat liver amounts to 5.0 ± 1.3 minutes, hereby differing statistically not significantly (p = 0.114) from the half-life of GLP-1 (7-37) with 6.1 ± 1.6 minutes.

Proteolytische Prozesse spielen eine wichtige Rolle während der Biogenese von Mitochondrien und bei der Qualitätskontrolle mitochondrialer Proteine. In der vorliegenden Arbeit wurde der Abbau von Proteinen in der Innen- und Außenmembran von Mitochondrien aus Saccharomyces cerevisiae untersucht. Ein erster Teil dieser Arbeit beschäftigt sich mit Mechanismen der Proteolyse in der mitochondrialen Innenmembran. Dazu wurde der Abbau einer mutanten Variante des polytopischen Membranproteins Oxa1 verfolgt. Es zeigte sich, dass die m-AAA-Protease den Abbau von Oxa1ts vermittelt, während keine Hinweise auf eine Beteiligung der i-AAAProtease erhalten wurden. In Abwesenheit der m-AAA-Protease wird Oxa1ts ebenfalls proteolytisch durch eine (oder mehrere) bislang nicht identifizierte Metallopeptidase(n) gespalten. Allerdings ist kein vollständiger Abbau von Oxa1ts durch diese Peptidase(n) zu beobachten. Vielmehr akkumulieren proteolytische Intermediate in den Mitochondrien. Nach den vorliegenden Untersuchungen kann die endoproteolytische Aktivität der Metallopeptidase(n) entweder eine Vorraussetzung für die Proteolyse durch die m-AAAProtease sein oder aber einen Bestandteil eines molekularen Ersatzsystems zum Abbau von mitochondrialen Innenmembranproteinen in Abwesenheit der m-AAAProtease darstellen. Während der Proteolyse durch AAA-Proteasen wird eine Dislokation von Membranproteinen beobachtet. Daher wurde eine mögliche Beteiligung von Translokationsporen der Innenmembran an Abbauvorgängen untersucht. Eine Rolle der TIM17/23-Translokase konnte ausgeschlossen werden. Des weiteren konnte auch für die OXA1-Pore keine essentielle Bedeutung für die Dislokation von Membranproteinen während der Proteolyse nachgewiesen werden. Eine Inaktivierung der Proteine Mba1 und Pnt1, die an Insertion von mitochondrialen Proteinen in die Innenmembran beteiligt sind, führte jedoch zu einer Beeinträchtigung von Abbauprozessen in der Innenmembran. Diese Befunde weisen auf eine Rezeptor- oder Chaperon-Funktion von Mba1 und Pnt1 während des Abbaus durch die AAA-Proteasen hin. In einem zweiten Teil der vorliegenden Arbeit wurde genetisch und biochemisch nach Komponenten gesucht, die den Abbau von Proteinen der mitochondrialen Außenmembran vermitteln. Ein Fusionsprotein, HA-DHFRWT-Tom6, das eine lösliche entfaltete Domäne in das Cytoplasma exponiert und im TOM-Komplex assembliert ist, wurde als Modellsubstrat verwendet. Während in isolierten Mitochondrien kein Abbau stattfindet, unterliegt das Protein in vivo deutlicher Proteolyse. Dieser Prozess wurde als ATP-abhängig charakterisiert. Eine Beteiligung des vakuolären Proteolyse-Systems, der i-AAA-Protease sowie des Ubiquitin- Proteasom-Abbauweges konnte unter den verwendeten experimentellen Bedingungen ausgeschlossen werden. Zur Identifizierung von Komponenten, die an der Proteolyse von Außenmembranproteinen beteiligt sind, wurde eine genetische Durchmusterung durchgeführt. Eine temperatursensitive Mutante des essentiellen Außenmembranproteins Tom40, das unter nicht-permissiven Bedingungen rasch abgebaut wird, wurde verwendet, um nach stabilisierenden Mutanten zu suchen. Die identifizierten Mutanten unterdrückten zwar den Wachstumsdefekt, führten aber zu keiner Stabilisierung von Tom40ts, weshalb keine am Abbau beteiligten Komponenten identifiziert werden konnten. Allerdings wurde durch die Isolierung eines Suppressors ein Bereich innerhalb des Proteins Tom40 beschrieben, der für die Assemblierung von Tom40 in den TOM-Komplex essentiell ist.

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH- terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping.

The mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP) cooperate in the proteolytic cleavage of matrix targeting sequences from nuclear-encoded mitochondrial precursor proteins. We have determined the cDNA sequence of Neurospora MPP after expression cloning. MPP appears to contain two domains of approximately equal size which are separated by a loop-like sequence. Considerable structural similarity exists to the recently sequenced yeast MPP as well as to Neurospora and yeast PEP. Four cysteine residues are conserved in Neurospora and yeast MPP. Inactivation of MPP can be achieved by using sulfhydryl reagents. MPP (but not PEP) depends on the presence of divalent metal ions for activity. Both MPP and PEP are synthesized as precursors containing matrix targeting signals which are processed during import into mitochondria by the mature forms of MPP and PEP.

Two proteins co-operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.

Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: (a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position-31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.