Mitochondrial preproteins as markers for Alzheimer's disease
10718784 · 2020-07-21
Assignee
Inventors
- Chris Meisinger (Freiburg, DE)
- Nora Vögtle (Freiburg, DE)
- Dirk Mossmann (Freiburg, DE)
- Elzbieta Glaser (Täby, SE)
- Caroline Graff (Sollentuna, SE)
- René P. Zahedi (Dortmund, DE)
Cpc classification
G01N2500/04
PHYSICS
G01N33/6842
PHYSICS
International classification
Abstract
The present invention is inter alia concerned with a method of diagnosing Alzheimer's disease in a patient, wherein said method is based on determining the amount of at least one premature mitochondrial protein. Further, the present invention relates to the use of such a protein as marker for Alzheimer's disease. Accordingly, antibodies binding to such a preprotein may be used for diagnosing Alzheimer's disease. The present invention is based on the finding that premature mitochondrial proteins accumulate in Alzheimer's disease.
Claims
1. A method of detecting an amount of at least one premature mitochondrial protein in a sample, the method comprising the following steps: a) providing a sample from a patient potentially suffering from Alzheimer's disease (AD); and b) detecting an amount of at least one premature mitochondrial protein in said sample, wherein said premature mitochondrial protein comprises at least part of its mitochondrium-targeting presequence, wherein the amount of said at least one premature mitochondrial protein is determined by a mass-spectrometry method, wherein said mass-spectrometry method selectively detects the at least one premature mitochondrial protein comprising at least part of its mitochondrium targeting presequence via the at least part of its mitochondrium-targeting presequence.
2. The method according to claim 1, wherein said sample is a blood sample or a brain sample.
3. The method according to claim 1, wherein said at least one premature mitochondrial protein is the protein mitochondrial human malate dehydrogenase 2 (hMdh2).
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(14) The present invention is based on the surprising finding that the mitochondrial protease MPP is functionally coupled to the mitochondrial protease PreP such that an inhibition of PreP by A leads to the inhibition of MPP. By consequence, mitochondrial precursor and intermediate precursor proteins accumulate. Thus, increased amounts of mitochondrial precursor and intermediate precursor proteins are indicative for the presence of A in mitochondria, which is characteristic for AD, particularly for an early stage of AD.
(15) Before the present invention is described in more detail, the following definitions are introduced.
1. Definitions
(16) As used in the specification and the claims, the singular forms of a and an also include the corresponding plurals unless the context clearly dictates otherwise.
(17) The term about in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of 10% and preferably 5%.
(18) It needs to be understood that the term comprising is not limiting. For the purposes of the present invention, the term consisting of is considered to be a preferred embodiment of the term comprising. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only. Likewise, if an isolated polypeptide is defined as comprising a specific sequence, this is also meant to encompass an isolated polypeptide which preferably consists of this specific sequence.
(19) Unless defined otherwise, all technical and scientific terms used herein have the meanings as commonly understood by a skilled person. Thus, e.g. the term Alzheimer's disease or AD as used herein refers to the disease including all symptoms (particularly dementia) as known to the skilled person.
(20) The term diagnosing AD in a patient as used herein means that the presence or absence of AD in a patient is determined. Thus, if the method of the present invention indicates a higher amount of said at least one premature mitochondrial protein in the sample from a patient potentially suffering from AD compared to the amount of said at least one premature mitochondrial protein in the control sample, the diagnosis of AD in said patient is positive (i.e. AD is present). If the method of the present invention fails to indicate a higher amount of said at least one premature mitochondrial protein in the sample from a patient potentially suffering from AD compared to the amount of said at least one premature mitochondrial protein in the control sample, the diagnosis of AD in said patient is negative (i.e. AD is not present). Diagnosing AD is thus not necessarily connected to a positive diagnosis.
(21) The term patient as used herein refers to a human or veterinary subject. Furthermore, the term includes both, living and dead patients.
(22) As used herein, the term sample refers to any biological sample from any human or veterinary subject. The samples may include tissues obtained from any organ, such as e.g. the brain and skin, and fluids obtained from any organ such as e.g. the blood, plasma, serum, lymphatic fluid, synovial fluid, cerebrospinal fluid, amniotic fluid, amniotic cord blood, tears, saliva, and nasopharyngeal washes. A brain sample, a skin sample, a blood sample and a cerebrospinal fluid sample are particularly preferred for the present invention. As noted above, a brain sample may particularly be provided if the patient is dead, whereas a blood sample may particularly be provided if the patient is alive. It is noted that a tissue sample (e.g. from the respiratory trast, the gastrointestinal tract, the skin or a muscle) of a living patient may of course also be provided. As regards samples differing from a brain sample and in particular as regards a blood sample, it is noted that mitochondrial dysfunctions in AD have not only been described for cells from the brain but also for peripheral cells, in particular blood cells comprising e.g. lymphocytes (see Leuner et al., 2012).
(23) If reference is made to a clinical sample, this indicates that the sample is handled according to standard proceedings used for samples in a clinical background, e.g. in hospitals or a medical practice.
(24) The term potentially suffering from AD as used in the present invention means that a patient has not yet been positively diagnosed with AD, e.g. by a histological analysis of brain tissue post mortem. The patient potentially suffering from AD may be nave with respect to initial observations as regards AD or may be a patient, who is already generally suspicious of suffering from AD, e.g. from a corresponding AD history of relatives, from cognitive tests or from imaging methods.
(25) The term determining the amount as used herein means that the amount of at least one protein is determined in relation to a second parameter such as e.g. the volume of a sample or the amount of cells in the sample or the presence of a different protein used as internal standard. This means that the amount is always normalized to a second parameter. Thus, the amount may e.g. correspond to a concentration if the second parameter is the volume. It is to be understood that the amount of the at least one protein determined in the control sample is also normalized to a second parameter, e.g. the parameters set out above. The skilled person understands that a comparison step as described herein is only possible if both amounts are normalized. The term determining the amount does not exclude that no such protein at all can be detected in a samplethis particularly applies to a protein, the amount of which is determined in a control sample. Thus, a protein found in a sample from a patient potentially suffering from AD (the amount of which is determined) may not be present at all in a control sample.
(26) For the present application, the term mitochondrial protein in particular relates to any nucleus-encoded protein, the gene of which is transcribed into mRNA in the nucleus, followed by the translation of the mRNA in the ribosome and subsequent release into the cytosol and the import into the mitochondrium. However, also mitochondrium-encoded proteins with a targeting sequence are encompassed by this term. Thus, a mitochondrial protein initially comprises a mitochondrium-targeting sequence. This sequence is normally cleaved off after successful import into a mitochondrium and/or a specific compartment therein, respectively. As used herein, the term does not relate to proteins, which completely lack any type of mitochondrium-targeting sequence.
(27) The term premature used in combination with mitochondrial protein means that the amino acid sequence does not correspond to the amino acid sequence of the mature mitochondrial protein; thus, at least part of the mitochondrium-targeting sequence is still present at the N-terminus of the mature sequence of the protein. It needs to be understood that premature as used herein does not mean that the complete mitochondrium-targeting sequence needs to be present at the N-terminus of the mature sequence of the protein. For this reason, several premature forms of a single mitochondrial protein may be present, wherein each form may comprise a mitochondrium-targeting sequence of a different length.
(28) The term mitochondrium-targeting presequence as used herein relates to a targeting sequence for nucleus-encoded mitochondrial proteins or for mitochondrium-encoded mitochondrial proteins, wherein said targeting sequence is found at the N-termini of said mitochondrial proteins after translation and prior to/during import into a mitochondrium or a compartment thereof, respectively. The targeting sequence for nucleus-encoded mitochondrial proteins is usually between about 10 and about 80 amino acids in length and it is assumed that the targeting sequence forms an amphiphatic -helix, in which positively charged amino acid side chains are located at one side of the helix, whereas uncharged polar amino acid side chains are located at the other side of the helix. It is emphasized that there is no unique mitochondrium-targeting sequence, which is found in exactly this sequence at the N-termini of all proteins targeted to the mitochondria. Rather, the above mentioned functional aspects of the sequence appear to be essential such that no unique order of amino acids appears to be required. For this reason, it is e.g. not possible to detect all premature mitochondrial proteins by a single antibody.
(29) The term at least part of the mitochondrium-targeting (pre)sequence means that said sequence has not been completely removed. Thus, said sequence can still be comprised as complete sequence (see above) or lack at least 1, 2, 3, 4, 5, 6, 7 or 8 amino acids from the N-terminus of the complete presequence. Depending on the length of the presequence, of course also more than 8 amino acids can be removed by initial cleavage, wherein a part of this presequence would then still be present.
(30) The term comparing as used herein means that the amount determined in step b) is compared to an amount derived from a control sample, wherein the amount of the control sample (control amount) must not necessarily be determined in parallel. The control amount may also be derived from a list comprising at least one predetermined value, which has been obtained from previous determinations with at least one control sample, preferably with several control samples, such as e.g. 10, 50, 100, 1000 or 10000 control samples. It can be preferred to obtain such predetermined control values from subjects not suffering from AD in connection with at least one further parameter, such as e.g. the age and/or the sex; if age-dependent predetermined control values are at hand, the age of the patient potentially suffering from AD may be aligned with a predetermined control value derived from subjects of the same age. It can be preferred to determine control values starting from the age of 40 years. The above comments regarding a normalization also apply to the control.
(31) The term higher amount as used herein means that the amount of the at least one premature mitochondrial protein in the sample from the patient potentially suffering from AD is increased compared to the amount of said at least one premature mitochondrial protein in the control sample by more than 5%, preferably by more than 10%, more preferably by more than 20% and most preferably by more than 50%. If said premature mitochondrial protein should not be detectable at all in the control sample, the presence of said premature mitochondrial protein in the sample from the patient potentially suffering from AD is indicative of AD. Thus, the term higher amount can also refer to a situation wherein said protein is present in the sample from a patient potentially suffering from AD and absent in the control sample.
(32) The term antibody as used herein preferably relates to a monoclonal or polyclonal antibody. However, the antibody may also be selected from antibody variants or fragments such as e.g. single chain antibodies, diabodies, minibodies, single chain Fv fragments (sc(Fv)), sc(Fv).sub.2 antibodies, Fab fragments, F(ab).sub.2 fragments, or tandem bodies. Antibodies may be produced according to any suitable method known to the person skilled in the art. Polyclonal antibodies may e.g. be produced by immunization of animals with the antigen of choice, whereas monoclonal antibodies of defined specificity may e.g. be produced using the hybridoma technology developed by Khler and Milstein. It is noted that an antibody as used herein may also be functionally linked, e.g. comprise a detectable label. The term binding fragment thereof relates to a fragment of an antibody, wherein such a fragment is still capable of binding the antigen. Preferably, such a fragment thus still comprises the CDR-regions of the underlying antibody.
(33) The term peptide refers to a molecular chain of amino acids connected via peptide bonds. Polypeptides according to the definition may be synthetic polypeptides that may include naturally or non-naturally occurring amino acids. A fragment of a peptide lacks at least one amino acid of the given sequence of a peptide.
(34) The term treatment of AD or treating AD as used herein may also relate to an alleviation of said disease and includes the treatment or alleviation of symptoms of AD.
(35) The term compound as used herein relates to any molecule, the skilled person considers suitable for possibly achieving an effect in an assay system comprising a compromised mitochondrial PreP. Particularly, molecules potentially influencing enzymatic activities of mitochondrial proteins will be considered by the skilled person.
(36) The term assay system as used herein relates to a typical system used by the skilled person in screening assays. Thus, the assay system may be a fully reconstituted in vitro system, wherein all necessary components are provided in a suitable buffer. The system may also be only partly reconstituted and e.g. comprise specific recombinantly expressed proteins together with isolated mitochondria or mitochondrial extracts gained from in vitro cultured cells, such as mammalian cells or yeast cells. The assays system may also be a system employing living cells, such as e.g. mammalian cells or yeast cells. Exemplary assays are set forth in the example section of the present application. Finally, specific labels such as e.g. fluorescent labels or radioactive labels may be used in the assay system, e.g. to determine the amounts of proteins.
(37) The term compromised as used herein relates to an at least partly inactive enzyme, in the present case PreP, which results in an accumulation of at least one premature mitochondrial protein. It is understood that the term PreP is used when referring to the human enzymeif e.g. yeast cells are used, the corresponding yeast homolog (Cym1) is compromised such that at least one premature mitochondrial protein accumulates.
(38) The term reactive oxygen species or ROS relates to chemically reactive molecules containing oxygen, which increase if mitochondria are compromised. Therefore, ROS can inter alia indicate the condition of mitochondria in cells.
(39) The term small molecule as used herein refers to a small organic compound having a low molecular weight. A small molecule may be a synthetic compound not known to occur in nature or a naturally-occurring compound isolated from or known to occur in natural sources, such as e.g. cells, plants, fungi, animals and the like. A small molecule in the context of the present invention preferably has a molecular weight of less than 5000 Dalton, more preferably of less than 4000 Dalton, more preferably less than 3000 Dalton, more preferably less than 2000 Dalton or even more preferably less than 1000 Dalton. In a particularly preferred embodiment a small molecule in the context of the present invention has a molecular weight of less than 800 Dalton. In another preferred embodiment, a small molecule in the context of the present invention has a molecular weight of 50 to 3000 Dalton, preferably of 100 to 2000 Dalton, more preferably of 100 to 1500 Dalton and even more preferably of 100 to 1000 Dalton. Most preferably, a small molecule in the context of the present invention has a molecular weight of 100 to 800 Dalton. It is further preferred that a small molecule in the context of the present invention meets the Rule of Five as set out below and is thus orally active (i.e. has a good oral bioavailability); these rules are as follows: the small molecule has no more than five hydrogen bond donors (e.g. nitrogen or oxygen atoms with one or more hydrogen atoms); the small molecule has not more than ten hydrogen bond acceptors (e.g. nitrogen or oxygen atoms); the small molecule has a molecular mass of less than 500 Dalton; the small molecule has an octanol-water partition coefficient log P not greater than 5.
2. Detailed Description of Certain Aspects of the Present Invention
2.1. Underlying Finding Derived from Results Shown in the Example Section
(40) The present invention is based on the surprising finding that the mitochondrial protease MPP (which is responsible for cleavage of N-terminal import presequences from nuclear-encoded mitochondrial proteins (21)) is functionally coupled to the protease PreP (which is responsible for degradation of the N-terminal import presequences).
(41) This functional link has the following implications for AD: It is known that the amyloid beta (A) protein is present in patients suffering from AD and that A is inter alia targeted to mitochondria within cells; further, it is known that A slows down/inhibits the activity of PreP (17). The functional link as found by the inventors results therein that MPP is also slowed down/inhibited. As a consequence, mitochondrial precursor proteins (still comprising N-terminal import presequences) and intermediate mitochondrial precursor proteins (still comprising parts of the N-terminal import presequences) accumulate. In consequence, mitochondrial functions are strongly impaired, e.g. with respect to respiration and the oxidative stress response. As regards the above-mentioned intermediate mitochondrial precursor proteins, it is noted that MPP not only catalyzes the complete cleavage of import presequences (resulting in the mature protein) but in some cases also only cleaves part of the import presequences (resulting in intermediate precursor proteins). Generally, such intermediate precursor proteins are further processed by Oct1/MIP (the octapeptidyl peptidase), which is also slowed down/inhibited if PreP is slowed down/inhibited.
(42) It is noted that mitochondrial dysfunctions including impaired cellular respiration, oxidative stress response, ATP synthesis, mtDNA maintenance and gene expression have been observed at early stages of AD and it has been proposed that mitochondrial dysfunction may serve as peripheral marker for the detection of AD in blood cells, especially in lymphocytes. It has also been proposed that early impairments of mitochondrial dysfunction and oxidative stress may precede A overproduction and deposition (so called mitochondrial cascade hypothesis). However, this has not yet been linked to an accumulation of mitochondrial precursor and intermediate precursor proteins.
(43) Due to the link found by the inventors, increased amounts of mitochondrial precursor and intermediate precursor proteins are indicative of the presence and/or an increased amount of A in mitochondria and cells, respectively. Increasing amounts of mitochondrial precursor and intermediate precursor proteins are therefore characteristic for AD and particularly for the early stage of AD.
2.2. Processing of Samples
(44) It can be preferred to process the sample provided in step a) of a method of the first aspect of the present invention prior to carrying out any further step(s).
(45) The processing step(s) inter alia depend on the method to be used for the determination of the amount carried out in step b) (including the determination of the presence/absence of at least one premature mitochondrial protein). Thus, if e.g. a Western-blot is used as immunological method, the final sample to be analyzed is typically provided in a denaturing buffer. For MS-analysis, different buffers known to the skilled person are available and are used in accordance with routine proceedings. Exemplary preparations, buffers and the like depending on the method to be used are given in the example section of the present application.
(46) Further, the processing step(s) also depend on the sample used in the method. If a tissue sample and in particular a brain sample (e.g. a sample from the temporal cortex) is used, the tissue is typically homogenized in order to lyse cells; a next step may be a centrifugation or the like to remove unbroken cells and nuclei. Typically, the mitochondria are then collected from the lysate by another centrifugation and lysed using e.g. a suitable lysis buffer. The protein content of this mitochondrial lysate is then typically analyzed in the subsequent step, wherein common methods such as determination of the concentration etc. may be used for standardization reasons. An exemplary processing of human brain samples is given in the example section of the present application.
(47) If blood as body fluid is used as sample, it may be necessary to concentrate mitochondria-containing cells; thus, the separation of erythrocytes can be preferred in order to remove cells, which do not contain any mitochondriastandard procedures known to the skilled person may be used for the separation of erythrocytes. It can therefore be preferred to collect and concentrate leukocytes and thrombocytes, followed by a lysis of said cells according to standard methods. The mitochondria may then be collected and lysed. The cells may also be directly used. As noted above for a brain sample, the protein content of the mitochondrial lysate is then typically analyzed in the subsequent step, wherein common methods such as determination of the concentration etc. may be used for standardization reasons. However, one may also use the supernatant of a body fluid sample (in particular blood) directly after an initial centrifugation step or even the body fluid sample itself, wherein a concentration step of the supernatant and sample, respectively (optionally carried out by ultrafiltration) might be required in order to concentrate the mitochondrial preproteins to be detected. It is noted that Example 3.3 below describes an exemplary way of using blood as sample.
(48) If skin is used as sample, it is preferred to use the fibroblasts comprised therein as cells underlying the analysis. In order obtain a sufficient quantity of fibroblasts from a skin sample, it may be necessary to isolate the fibroblasts and/or concentrate the fibroblasts. It is preferred to cultivate the fibroblasts according to standard methods prior to carrying out the analysis in order to increase the fibroblast cell number. The use of skin fibroblasts and such standard methods have been described earlier, e.g. in WO 02/067764 (see in particular the section Processing and culture of fibroblasts from fresh biopsies on pages 22 and 23 of WO 02/067764).
(49) Typical protocols and buffers are known to the skilled person and can e.g. be found on the following two homepages: embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/en.wikipedia.org/wiki/Lysis_buffer
(50) Thus, a method of the present invention including the above mentioned additional steps may also be formulated as follows:
(51) Method of diagnosing Alzheimer's disease (AD) in a patient comprising the following steps: Providing a sample from a patient potentially suffering from AD; Collecting and/or concentration cells containing mitochondria from said sample; Lysing said cells and optionally collecting and/or concentrating mitochondria; Lysing said cells/mitochondria; Determining the amount of at least one premature mitochondrial protein, wherein said premature mitochondrial protein comprises at least part of its mitochondrium-targeting presequence; and Comparing the amount obtained in the previous step to the amount of said at least one premature mitochondrial protein determined in a control sample, wherein said control sample is derived from a subject not suffering from AD;
wherein a higher amount of said at least one premature mitochondrial protein in the sample from a patient potentially suffering from AD compared to the amount of said at least one premature mitochondrial protein in the control sample indicates AD in said patient.
2.3. Preferred Precursor Proteins
(52) Precursor and intermediate precursor forms of the following mitochondrial proteins are particularly preferred in the present invention (wherein all isoforms and splice variants are also included); in the following, not only details about these proteins but also their mitochondrium-targeting presequences will be given (starting from the N-terminus):
(53) TABLE-US-00001 hMdh2:Humanmalatdehydrogenase2,mitochondrial [MDH2human,GenBankacc.no.CAG38785.1] (SEQIDNo.:20) MLSALARPASAALRRSFSTSAQNN hOAT:Humanornithinaminotransferase, mitochondrial [OAThuman,UniProtKBacc.no.P04181] (SEQIDNo.:21) MFSKLAHLQRFAVLSRGVHSSVASATSVATKKTVQ hACADV:Humanverylong-chainspecificacyl-CoA dehydrogenase,mitochondrial [ACADVhuman,UniProtKBacc.no.P49748] (SEQIDNo.:22) MQAARMAASLGRQLLRLGGGSSRLTALLGQPRPGPARRPY PMPCA:mitochondrial-processingpeptidasesubunit alpha,mitochondrial [UniProtKBacc.no.Q10713] (SEQIDNo.:23) MAAVVLAATRLLRGSGSWGCSRLRFGPPAYRRF CLYBL:Citratelyasesubunitbeta-likeprotein, mitochondrial [UniProtKBacc.no.Q8N0X4] (SEQIDNo.:24) MALRLLRRAARGAAAAALLRLK PPM1K:Proteinph,mitochondrial [PPM1K,UniProtKBacc.no.Q8N3J5] (SEQIDNo.:25) MSTAALITLVRSGGNQVRRRVLLSSRLLQ SLIRP:Stem-loop-interactingRNA-bindingprotein, mitochondrial [SRA,UniProtKBacc.no.Q9GZT3] (SEQIDNo.:26) MAASAARGAAALRRSINQPVAFVRRIPW NDUFA9:NADHdehydrogenase[ubiquinone] 1alpha subcomplexsubunit9,mitochondrial [NDUA_human,UniProtKBacc.no.Q16795] (SEQIDNo.:69) MAAAAQSRVVRVLSMSRSAITAIATSVCHGPPCRQ MRPL23:39SribosomalproteinL23,mitochondrial [RM23_human,UniProtKBacc.no.Q16540] (SEQIDNo.:70) MARNVVYPLYRLGGPQLRVFRT
3. Examples
3.1. Example 1
(54) Mitochondrial dysfunction plays an important role in the pathology of Alzheimer's disease (AD). Although it is still unclear if mitochondrial dysfunction is cause or consequence of AD and how it is connected to other cellular dysfunctions (1-4), A appears to accumulate in mitochondria of AD patients and affects a multitude of functions including respiration, detoxification of reactive oxygen species (ROS) and organellar morphology (2, 5-14). A can be cleared by the mitochondrial matrix peptidasome PreP, a metallopeptidase that degrades peptides including presequence peptides generated upon maturation of imported precursor proteins and that has a decreased activity in AD (15-17). Many mitochondrial proteins contain N-terminal presequences that direct these precursors from the cytosol into the organelle. Upon import, presequences are cleaved by the mitochondrial processing peptidase MPP in the matrix releasing the mature protein (18-21). In several cases MPP generates intermediate forms that are further processed by the octapeptidyl peptidase Oct1/MIP or the intermediate cleaving peptidase Icp55.
(55) Isolated mitochondria from post mortem brain samples of four AD patients and four age-matched non-AD controls (table S2) were analyzed and the presence of higher molecular precursor species of the matrix protein MDH2 in all patient samples but not in controls was observed (
(56) To uncover a functional link between mitochondrial preprotein maturation and peptide turnover and its potential role in A toxicity, S. cerevisiae that represents an ideal model organism to study basic mechanisms underlying human diseases including AD was used (3, 25). A mutant was generated that lacks the yeast PreP homolog Cym1 (26) and harbors a temperature-sensitive allele of the essential MPP subunit Mas1 (18, 19, 21). The mutant was not able to grow under respiratory conditions (i.e. conditions in which mitochondrial energy metabolism is essential for cell viability) indicating a genetic interaction of the presequence peptidase MPP and the peptidasome Cym1 (
(57) To directly analyze a dependence of preprotein maturation on peptide turnover an in vitro processing assay was employed in mitochondrial extracts (
(58) It was found that A peptides were degraded by Cym1 in mitochondrial extracts and by the recombinant enzyme (
(59) The next question was if mitochondrial A could impair precursor maturation and two model systems were chosen. Firstly, freshly prepared soluble extracts from brain mitochondria of PS2APP mice were tested. This AD model harbors mutations in the PS2 (N141I) and APP (Swedish FAD) genes and shows A accumulation in mitochondria (
3.2. Example 2
(60) The aim of this example was to detect and determine human mitochondrial proteins comprising at least part of their N-terminal presequence (i.e. preproteins) in brain samples from AD patients compared to age-matched controls without AD.
(61) The samples were analyzed according to the CHAFRADIC method described in Venne et al., J. Proteome Res. 12, 3823 (2013). Human mitochondrial samples (see section 3.3. below for details) were lysed with lysis buffer (2% SDS, 150 mM NaCl, 50 mM Tris, pH 7.8) and subsequently carbamido-methylated. Thus, the lysates were initially reduced with 10 mM DTT for 30 minutes at 56 C. and then alkylated with 20 mM IAA for 30 minutes at room temperature in the dark.
(62) For the specific dimethyl-labeling of the free protein N-termini and lysine-residues, 100 g of each of the control and AD-sample were treated with a light and heavy labeling, respectively, for 2 hours at 37 C. according to the protocol by Jentoft et al., J Biol Chem 1979, 254(11):4359-4365. For conducting the light labeling, the control sample was incubated with 20 mM CH.sub.2O, 40 mM NaBH.sub.3CN in 200 mM HEPES, pH 8.0. The AD sample was labeled with a heavy label by using 20 mM CD.sub.2O, 40 mM NaBD.sub.3CN in 200 mM HEPES. The reaction was blocked via incubation with 60 mM glycine for 10 minutes and 130 mM hydroxylamine for 15 minutes at room temperature. The samples were then pooled in a ratio of 1:1 and an ethanol precipitation was carried out.
(63) The pooled sample was treated with ice-cold ethanol in a ratio of 1:10 and incubated for 1 hour at 40 C., followed by pelleting the proteins via centrifugation at 4 C. for 30 minutes. After discharging the supernatant, the pellet was dried initially at room temperature and then solubilized in 40 L 2M GuHCl, 50 mM Na.sub.2HPO.sub.4, pH 7.8. The protein solution was then diluted for the subsequent proteolytic digestion with digestion buffer (50 mM NH.sub.4HCO.sub.3, 5% Acetonitril, 1 mM CaCl.sub.2, pH 7.8) in a ratio of 1:10. The proteolytic digestion was carried out using trypsin from Promega in a ratio of 1:30 for 12 hours at 37 C.
(64) After a monolithic digestion control was carried out (Burkhart J M et al-. J Proteomics 2012; 75(4):1454-1462), the sample was prepared for the strong cation exchange, SCX. The sample was desaltet using a 4 mg C18 SPEC cartridge. SCX separation was carried out on a U3000 HPLC system (Thermo Scientific) in combination with a 1501 mm POLYSULFOETHYL A column (PolyLC, Columbia, US, 5 m particle diameter, 200 A pore size) using three buffers: SCX buffer A (10 mM KH.sub.2PO.sub.4, 20% ACN, pH 2.7), SCX buffer B (10 mM KH.sub.2PO4, 188 KCl, 20% ACN, pH 2.7) and SCX buffer C (10 mM KH.sub.2PO.sub.4, 800 mM NaCl, 20% ACN, pH 2.7). 100 g of the desalted sample were resuspended in 50 l SCX buffer A and separated at a flow of 80 g/ml with a gradient optimized for the peptide charge conditions: over 10 minutes, 100% SCX buffer A was used; then, the ratio of SCX buffer B increased over 18 minutes from 0 to 20%, wherein the gradient was maintained for 10 minutes at 20% SCX buffer B. Then, the ratio of SCX buffer B was increased within 2 minutes in a linear way to 40% B and maintained for further 5 minutes, before the ratio of SCX buffer B was increased in a linear way within 5 minutes to 100%. After 5 minutes at 100% SCX buffer B, the column was washed for 5 minutes with 100% SCX buffer C. Fractions of the charge conditions +1, +2, +3, +4, +5 were automatically collected and concentrated to a volume of 40 l employing vacuum.
(65) For chemical derivatization of the N-termini of internal peptides, the fractions were adjusted to a final volume of 300 l with 200 mM Na.sub.2HPO.sub.4, pH 8.0 and then in two steps incubated for 1 hour each with initially 20 mM and then 10 mM NHS-trisdeuteroacetate at 37 C. (Staes A et al., Nat Protoc 2011, 6(8):1130-1141). The reaction was blocked via incubation with 60 mM glycine for 10 minutes and 130 mM hydroxylamine for 15 minutes at room temperature. The fractions were then desalted as described above using 4 mg C18 SPEC cartridges (Agilent) and after concentrating the samples to the dry state dissolved in 50 l SCX buffer A. Rechromatography of the charge conditions+1, +2, +3, +4, +5 took place under the conditions described above in independent separations, wherein again the corresponding charge conditions were collected. They were then desalted as described above using 4 mg C18 SPEC cartiladges (Agilent) and after concentration to the dry sate resuspended under vacuum in 15 l 0.1% trifluoroacetic acid (TFA).
(66) The quantitative analysis of the concentrated N-terminal peptides was carried out using LC-MS and the results are given in the following table:
(67) Identification of mitochondrial N-termini of mitochondrial precursor proteins from human brain mitochondria (temporal lobe); peptide ratios are given in AD/controls; grey: accumulated presequence peptides.
3.3. Detection of Premature MDH2 in Blood
(68) TABLE-US-00002 PATIENT 2 PATIENT 1 median median AD/control AD/control times ratio and times ratio and identified quantified corresponding quantified corresponding Pre- N-terminal (quan) and standard (quan) and standard sequence peptide identified deviation identified deviation acc. to First Last (PSMs) MD SD (PSMs) MD SD Acc. Protein Uniprot AA AA Sequ #quan #PSMs ratio ratio #quan #PSMs ratio ratio P04181 OAT_HUMAN 1 to 36 26 46 tSVATk 3 7 2.31 0.10 4 8 2.87 0.29 Ornithine kTVQG aminotransferase, PPTSD mitochondrial DIFER P49748 ACADV_HUMAN 1 to 40 7 16 aASLG 11 37 100.00 36.73 Very long-chain pred. RQLLR specific acyl-CoA dehydrogenase, mitochondrial Q10713 Mitochondrial- 1 to 33 2 10 aAVVL 1 2 8.91 2 6 6.76 3.66 processing AATR peptidase subunit alpha Q8NOX4 Citrate lyase 1 to 22 9 20 aARGA 4 5 100.00 subunit beta- pred. AAAAL like protein, LR mitochondrial Q8N3J5 PPM1K_HUMAN 1 to 29 4 11 aALItL 9 17 100.00 4 6 77.02 26.74 Protein ph VR Q9GZT3 SRA stem-loop- 1 to 28 4 13 sAARG 4 13 100.00 1 3 100.00 interacting pred. AAALR RNA-binding protein, mitochondrial
(69) Fresh blood samples were taken from two patients diagnosed with AD and two age-matched human subjects not suffering from AD (referred to as controls in the following).
(70) Peripheral blood mononuclear cells (PBMCs) were isolated from the samples and cryopreserved according to reference 43. Briefly, PBMC were isolated from fresh EDTA blood, diluted with the same volume of Ca.sup.2+Mg.sup.2+ free Hanks balanced salt solution (HBSS from PAA) or phosphate buffer saline (PBS from PAA) and pipetted carefully over Ficoll-Hypaque (Linaris) gradients in Falcon tubes with 1:2 ratio of Ficoll-Hypaque to diluted blood. After centrifugation (25-30 minutes, 810 g, no brake, room temperature) the interface with the PBMCs was collected. Cells were washed three times with HBSS (10 min, 300 g, room temperature) and counted with a hemocytometer using trypan blue (Sigma) to discriminate between living and dead cells. For freezing, cells were resuspended cautiously with 40% foetal calf serum (FBS from Sigma or FCS from Invitrogen) in RPMI 1640 (Gibco) at room temperature. The same volume of 20% DMSO (Serva) in RPMI was added in two steps with 5 min waiting in between. Cells in special cryovials (Greiner) were placed into cardboard boxes and moved immediately to a 80 C. freezer and to liquid nitrogen for long term storage.
(71) Subsequently, the PBMCs were further fractionated in the monocytes and the non-monocytes fractions.
(72) In order to separate the PBMCs into these two fractions, the MACS technology by Miltenyi was used. Following the manufacturer's (Miltenyi, Bergisch Gladbach, Germany) instructions, PBMCs were incubated with FC receptor blocker (provided by the manufacturer) to block unspecific antibody binding, washed and incubated with special monoclonal antibodies which target specific surface molecules on the desired cell population (in the present case CD14 for monocyte isolation) or in case of negative selection on the rest of the PBMCs (i.e. non-monocytes). These antibodies are linked to super paramagnetic particles (MACS MicroBeads). When the cell suspension was pipetted onto columns (provided by the manufacturer) on which a strong magnetic field is applied the labelled cells were retained. Through simple rinsing the non-labelled cells were collected. After detaching the column from the magnet also the labelled cell fraction was rinsed, collected and used for further experiments. With a hemocytometer (Neubauer chamber) the yield of each fraction was checked and the purity and success of the separation was confirmed by flow cytometry according to standard methods.
(73) Following the above procedure, (i) monocytes and (ii) non-monocytes fractions derived from PBMC were available from two AD patients and two controls.
(74) Samples of these fractions were separated by a denaturing SDS-PAGE (a protein amount of about 10 g was used for each sample [and thus per lane]) according to standard methods, and a Western-Blot was carried out according to standard methods using the afore-mentioned antibody recognizing only precursor MDH2 (but not the mature cleaved protein, see also experimental procedures described in the following example). As can be derived from
3.4. Experimental Procedures Used in the Above Examples
(75) Yeast Strains and Growth Conditions
(76) Yeast strains used in this study are listed in table S1. Yeast cells were grown on YPD medium (1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) glucose) or YPG medium (containing 3% (w/v) glycerol instead of glucose). Deletion mutants were generated by homologous recombination (31). For growth tests yeast cells were cultured in 5 ml YPD medium at 30 C. Cell numbers (OD.sub.600) were adjusted and 5-fold serial dilutions were spotted on YPD or YPG agar plates.
(77) Isolation of Mitochondria from Yeast
(78) Yeast cells were grown at 24 C. (if not indicated otherwise) to an OD.sub.600 of 1.0-1.5. Cells were harvested, washed in dH.sub.2O and incubated in DTT buffer (0.1 M Tris/H.sub.2SO.sub.4, pH 9.4, 10 mM DTT) for 20 min. After re-isolation cell pellets were resuspended in zymolyase buffer (1.2 M sorbitol, 20 mM K.sub.2HPO.sub.4/HCl, pH 7.4) supplemented with 3 mg/mg (wet weight cells) zymolyase and incubated for 40 min at 24 C. Resulting spheroblasts were washed in zymolyase buffer without enzyme and resuspended in homogenizing buffer (0.6 M sorbitol, 10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 0.2% (w/v) BSA, 1 mM PMSF). Cells were subjected to 20 strokes in a glass homogenizer. Cellular debris was removed by two consecutive centrifugation steps for 5 min at 1500g at 4 C. Mitochondria were isolated by centrifugation for 15 min at 16,000g at 4 C. Mitochondria were suspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS/KOH, pH 7.2) and protein concentration was determined by Bradford assay and adjusted to 10 mg/ml. Aliquots were snap-frozen in liquid nitrogen and stored at 80 C.
(79) Processing and Degradation Assays Using Yeast Soluble Mitochondrial Extracts
(80) Isolated mitochondria from yeast strains grown under respiratory conditions were washed with SEM buffer, re-isolated and resuspended in reaction buffer (250 mM sucrose, 10 mM MOPS/KOH, pH 7.2, 80 mM KCl, 5 mM MgCl.sub.2, 5 mM KH.sub.2PO.sub.4) to a concentration of 3 g/l. Mitochondria were subjected to sonication (five times 30 s with 30 s breaks on ice, Sonifier250, Branson) followed by centrifugation at 100,000g for 45 min at 4 C. The obtained supernatant was used for processing and degradation experiments. 0.5 l of radiolabelled precursor protein and/or various peptides in different concentrations were added to 9 l of yeast soluble mitochondrial extracts. Reactions were stopped by addition of 4 Laemmli buffer (8% (w/v) SDS, 0.08% (w/v) bromophenol blue, 40% (v/v) glycerol, 240 mM Tris/HCl, pH 6.8) containing 5% (v/v) -mercaptoethanol and analyzed by SDS-PAGE followed by autoradiography and immunodecoration. Radiolabelled precursor proteins were synthesized with the transcription/translation rabbit reticulate lysate system (Promega) in the presence of .sup.35S-methionine. Chemical amounts of Cym1 and control protein (Oct1) were synthesized in vitro using the RTS wheat germ system (5 PRIME). 0.5 l of cell-free translation product was used in the processing assays.
(81) Membrane Potential Measurement
(82) The membrane potential () was measured by fluorescence quenching. Isolated yeast mitochondria (50 g) were incubated in 3 ml potential buffer (0.6 M sorbitol, 0.1% (w/v) BSA, 10 mM MgCl.sub.2, 0.5 mM EDTA, 20 mM KPi, pH7.2) in the presence of 3 l DiSC3 (3,3-dipropylthiadicarbocyanine iodide, 2 mM in ethanol). Samples were mixed and absorption measured until a distribution equilibrium was reached (excitation 622 nm, emission 670 nm) using the luminescence spectrometer Aminco Bowman2 (Thermo Electron Corporation). The membrane potential was dissipated by addition of 4 l valinomycin (1 mM in ethanol). Data were analysed with FL WinLab (Perkin Elmer).
(83) High Resolution Respirometry
(84) Mitochondrial respiration was measured with the Oxygraph 2-k (Oroboros Instruments, Austria) and analyzed with the DATLAB software. Measurements were performed at 30 C. in a 2 ml chamber. Isolated yeast mitochondria (100 g) were added to 2 ml respiration buffer (10 mM MOPS/KOH, pH 7.2, 250 mM sucrose, 5 mM MgCl.sub.2, 80 mM KCl, 5 mM KP.sub.i) supplemented with 1 mM NADH and 1 mM ADP to obtain a basic respiration rate. The respiration rate was measured over a time-course of 5 min and the obtained wild-type values set to 100%.
(85) Detection of Reactive Oxygen Species
(86) For dihydroethidium (DHE) staining (10) 20 g yeast mitochondria were washed with SEM buffer and incubated with 1 M DHE in reaction buffer in the dark for 10 min. Fluorescence units were measured using a fluorescence reader (Infinite M200, Tecan) at an excitation wavelength of 480 nm and emission wavelength of 604 nm. Samples were measured in triplicates and background signals (samples without mitochondria) were subtracted. The wild-type values were set to 100%.
(87) Expression and Purification of Mitochondrial Processing Peptidase (MPP) Complex
(88) E. coli BL21 cells were transformed with plasmid pVG18 (32) that enables the transcription of a single mRNA encoding both - (with N-terminal poly-histidine tag) and -MPP subunits (Mas1 and Mas2) from S. cerevisiae. Cell cultures were grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) containing 100 g/ml ampicillin at 37 C. to an OD.sub.600 of 0.6. Expression was induced by addition of 1 mM IPTG (Isopropyl -D-1-thiogalactopyranoside, Formedium) for 4 h. Cells were harvested and snap-frozen in liquid nitrogen. Pellets were resuspended in binding buffer (20 mM Na.sub.3PO.sub.4, 200 mM NaCl, 20 mM imidazole, pH 7.4) containing 1 mg/ml lysozyme (Sigma) and 10 g/ml DNaseI (Sigma) and incubated for 1 h at 4 C. for cell lysis. The cell extract was centrifuged at 4000g to remove unbroken cells. The supernatant was further centrifuged at 100,000g for 30 min at 4 C. The obtained supernatant was incubated with Ni-NTA resin (Qiagen) for 3 h at 4 C. The resin was washed four times with binding buffer. Bound proteins were eluted with Elution buffer (20 mM Na3PO4, 200 mM NaCl, 200 mM imidazole, pH 7.4). The eluate was concentrated in a 50 kDa cut-off filter (Milipore) and applied to a Superdex 200 10/300 GL size-exclusion column equilibrated in 10 mM HEPES/KOH, pH 7.4, 50 mM NaCl, 1 mM DTT for further purification of the expressed protein.
(89) MPP Activity Assays
(90) Purified yeast MPP (0.15 g protein) was incubated in processing buffer (10 mM HEPES/KOH, pH 7.4, 0.1 mM MnCl.sub.2, 1 mM DTT, 0.1 mg/ml BSA) for 15 min at 30 C. (reaction volume of 20 l) with the indicated peptides (see Miscellaneous). After pre-incubation, 1 l of [.sup.35S]-labeled F.sub.1 precursor protein (Nicotiana plumbaginifolia) was added and incubated for 10 min at 30 C. The reactions were stopped by addition of SDS-sample buffer (150 mM Tris/HCl, pH 7.0, 12% (w/v) SDS, 6% (w/v)-mercaptoethanol, 30% (v/v) glycerol, 0.05% (w/v) bromophenol blue). The samples were analyzed by SDS-PAGE followed by autoradiography. Bands were quantified using the Multi Gauge software (Fuji Film). Experiments were performed in triplicates.
(91) Transgenic Mice
(92) The transgenic mouse line PS2APP (line B6.152H) is homozygous for both human PS2 N141I and APP Swedish FAD transgenes. (10, 27). As control wild-type age-matched C57Bl/6 mice were used. Mice were kept under an alternating cycle of 12-h light/12-h dark (lights were switched on at 6:00 am) at 20-22 C. Water and food was provided ad libitum. Strict adherence to the German federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care was maintained for an procedures and an experiments were carried out with the explicit approval of the local veterinary authority.
(93) Isolation of Mitochondria from Mouse Tissue
(94) One year old mice were sacrificed and brains aseptically removed from the skull. Brain stem and cerebellum were excised. An following steps were carried out on ice. Tissue was sliced with a scalpel and weighed. Samples were suspended in solution B (20 mM HEPES/KOH, pH 7.6, 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 0.5 mM PMSF) at approximately 10 ml/g brain tissue and homogenized by 30 strokes in a glass potter. Non-broken cells were removed by centrifugation at 800g for 15 min at 4 C. Supernatant was subjected to centrifugation at 7000g for 15 min at 4 C. for isolation of mitochondria. The mitochondrial pellet was resuspended in solution B and the protein concentration determined by Bradford protein assay. Samples were adjusted to 10 mg/ml with sucrose buffer (10 mM HEPES/KOH, pH 7.6, 0.5 M sucrose), aliquoted, snap-frozen in liquid nitrogen and stored at 80 C.
(95) Processing Assays Using Mouse Soluble Mitochondrial Extracts
(96) Isolated mitochondria were washed, re-isolated and solubilized in reaction buffer containing 1% (w/v) digitonin and 1 mM MnCl.sub.2. Samples were incubated on ice for 15 min. After centrifugation at 20,000g for 10 min at 4 C. the obtained supernatant was used for processing assays. Samples containing 60 g mitochondrial extract in reaction buffer were incubated with 0.5 l radiolabelled precursor protein at 37 C. for different times. The reactions were stopped by addition of 4 Laemmli buffer containing 5% (v/v) -mercaptoethanol. Samples were analysed by SDS-PAGE followed by autoradiography and immunodecoration.
(97) Organelle Isolation from Temporal Cortex of Human Brains
(98) The tissue samples (temporal cortex from human brains) used in this study originated from individuals diagnosed with Alzheimer's disease (AD) and age-matched control individuals without AD and were obtained from the Karolinska Institute, Stockholm (table S2). Samples were collected according to local regulations for diagnostic purposes. The tissue samples were anonymized after diagnostic evaluation and used for this study with approval of the Ethical Committee (Dnr 2011/962-31/1, Stockholm). The temporal cortex regions were homogenized with a glass/Teflon homogenizer (15 strokes) in Buffer A (230 mM Mannitol, 70 mM Sucrose, 20 mM HEPES/KOH, pH 7.2, 0.5 mM EDTA). Unbroken cells and nuclei were removed by centrifugation at 484g for 5 min at 4 C. In order to collect mitochondria the supernatant fraction was centrifuged at 7741g for 10 min at 4 C. Both centrifugation steps were repeated. The mitochondrial pellet was resuspended in buffer A and the protein concentration determined by Bradford assay.
(99) Expression of A in Yeast
(100) The plasmid encoding a cytosolically expressed TEV (Tobacco Etch Virus) protease (p416.sup.TEVcyt) was generated by deleting the b.sub.2-presequence of pRS416GAL1-b.sub.2-TEV (33) by PCR using primers 5-CGTCAAGGAGAAAAAACCCCGGATTCTAGCATGAGATCCAGCTTGTTTA AGGGACCACGTG-3 (SEQ ID No.: 1) and 5-CACGTGGTCCCTTAAACAAGCTGGATCTCATGCTA GAATCCGGGGTTTTTTCTCCTTGACG-3 (SEQ ID No.: 2). For generation of the pESC.sup.eGFP-A vector, eGFP was amplified by PCR, using pUG35(Ura) as template, with the primers 5-ATCTGAATTCATGTCTAAAGGTGAAGAATTATTCAC-3 (SEQ ID No.: 3) and 5-ATCTGAATTCTT TGTACAATTCATCCATACCATG-3 (SEQ ID No.: 4), digested with EcoRI and ligated into pESC(His) (Stratagene). This pESC-eGFP vector contained a linker (sequence: RIQPSLKGGRTS; according to the main part of the multiple cloning site (MCS)) to guarantee proper folding of eGFP and A.sup.1-42 and a stop codon in frame with the MCS. The cloning vector pESC-eGFP_G omitting the stop codon was created by PCR using primers 5-ATCTGAATTCATGTCTAAAGGTGAAGAATTATTCAC-3 (SEQ ID No.: 5) and 5-ATCTG AATTCGTTTGTACAATTCATCCATACCATG-3 (SEQ ID No.: 6). A.sup.1-42 was amplified by PCR with primers 5-ATCTACTAGTATGGATGCAGAATTCCGACATGAC-3 (Seq ID No.: 7) and 5-ATCTATCG ATTTACGCTATGACAACACCGCCC-3 (SEQ ID No.: 8) using pAS1N-A-GFP as template (34), digested with SpeI and ClaI and cloned into pESC-eGFP_G. A TEV cleavage site was inserted 5 to A.sup.1-42 by PCR using primers 5-GGGCGGCCGCACTAGTGAGAACCTGTACTTCCAGTCCGATGCAGAATTCC GACATGACTCAGG-3 (SEQ ID No.: 9) and 5-CGGAATTCTGCATCGGACTGGAAGTACAGGTTCTCACTAGTGCGGCCGCC CTTTAGTGAGGG-3 (SEQ ID No.: 10).
(101) Strains were transformed with p416.sup.Tevcyt and pESC.sup.eGFP-A or pESC.sup.ev (empty vector) as described above. Cells were grown in selective medium (6.7% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose and 0.77% (w/v) Complete Supplement Mixture lacking histidine and uracil) at 30 C. For induction of expression, cells were shifted to selective medium containing 2% (w/v) galactose instead of glucose and incubated at 30 C. for 1 or 3 days, respectively. Yeast cell extracts were generated by the post-alkaline extraction method (35). Briefly, 2.5 OD.sub.600 of yeast cells were washed with dH.sub.2O, resuspended in 0.1 M NaOH and incubated for 5 minutes at 25 C. with 1400 rpm shaking. After re-isolation, cells were resuspended in 1 Laemmli buffer containing 5% (v/v) -mercaptoethanol and analyzed by SDS-PAGE and immunodecoration.
(102) Enrichment of N-Terminal Peptides Using COFRADIC
(103) N-terminal COFRADIC was conducted as previously described with the following modifications (21, 36, 37). Highly pure mitochondria pellets (from yeast strains grown on YPG, 30 C.) were lysed in 500 l of 2 M guanidium hydrochloride, 50 mM sodium phosphate, pH 8.7. Disulfide bonds were reduced by addition of 10 mM dithiotreithol for 30 min in 56 C. and free sulfhydryl groups were subsequently carbamidomethylated using 20 mM iodoacetamide at 25 C. in the dark. Afterwards, lysine residues were acetylated by incubation with 25 mM deutero-acetyl N-hydroxy-succimide for 1 h at 37 C. The reaction was quenched by adding a 4-fold molar excess of hydroxylamine and a 2-fold excess of glycine. Samples were diluted 10-fold with 50 mM ammonium bicarbonate, 5% (v/v) acetonitrile (ACN), 1 mM CaCl.sub.2, pH 7.8 and digested with trypsin at 37 C. overnight (protease to protein ratio of 1:20). Generated peptides were purified by solid phase extraction with C18AR columns (Varian) according to the manufacturer's protocol and dried under vacuum. Peptides were reconstituted in 0.08% (v/v) trifluoroacetic acid (TFA), 50% (v/v) ACN, pH 2.7, and loaded onto a strong cation exchange (SCX) tip which was equilibrated with 10 mM Na.sub.3PO.sub.4, 50% (v/v) ACN, pH 2.7. Singly charged peptides were eluted with 10 mM Na.sub.3PO.sub.4, 50% (v/v) ACN, pH 2.7. This fraction was dried under vacuum and reconstituted in 100 l of 10 mM ammonium acetate, 2% (v/v) ACN, pH 5.5. H.sub.2O.sub.2 was added to a final concentration of 0.5% (v/v) and incubated at 30 C. for 30 min, immediately prior to the primary RP-HPLC separation. Peptides were separated on an Ultimate 3000 LC system (Thermo Scientific) equipped with an 8-port valve WPS-T 3000 well plate sampler using a Zorbax 300SB-C18 column (5 m particle size, 2.1150 mm, Agilent) at a flow rate of 80 l/min at 30 C. Solvent A was 10 mM ammonium acetate, 2% (v/v) ACN, pH 5.5, and solvent B was 10 mM ammonium acetate, 70% (v/v) ACN, pH 5.5. During the primary run 16 fractions of 4 min each were collected and subsequently dried under vacuum. Finally, peptides were reconstituted in 50 mM sodium borate, pH 9.5. For derivatization of free internal peptide N-termini 6 M 2,4,6-trinitrobenzenesulfonic acid (TNBS) was added to each fraction and incubated for 30 min at 37 C. This step was repeated thrice. The reaction was stopped by addition of TFA to a final pH of 2. Afterwards, derivatized fractions were applied to a secondary RP-HPLC run, using identical chromatographic conditions as above. This time, fractions were recollected in a time frame starting 4 min before and ending 4 min after the original elution time in the primary run. Fractions were dried under vacuum and prepared for LC-MS analysis by reconstitution in 0.1% (v/v) TFA.
(104) Nano-LC-MS/MS
(105) Samples were analyzed on an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific), online coupled to a U3000 nano-HPLC system (Thermo Scientific). Peptides were preconcentrated on an in-house packed 100 m inner diameter C18 trapping column (Synergi HydroRP, Phenomenex, 4 m particle size, 80 pore size, 2 cm length) in 0.1% trifluoroacetic acid and separated on an in-house packed 75 m inner diameter C18 main-column (Synergi HydroRP, Phenomenex, 2 m particle size, 80 pore size, 30 cm length) applying a binary gradient from 4-42% acetonitrile in 0.1% formic acid. Dedicated wash blanks were introduced between consecutive samples to eliminate memory effects (38). MS survey scans were acquired in the Orbitrap from m/z 300 to 2000 at a resolution of 60,000 using the polysiloxane m/z 445.120030 as lock mass. The ten most intense signals were subjected to collision induced dissociation (CID) in the ion trap, taking into account a dynamic exclusion of 30 s. CID spectra were acquired with a normalized CE of 35%, an isolation width of 2 m/z, an activation time of 30 ms and a maximum injection time of 100 ms. Automatic gain control (AGC) target values were set to 10.sup.6 for MS and 10.sup.4 for MS/MS scans. Data interpretation was accomplished as previously described in (21).
(106) Miscellaneous
(107) Rabbits were immunized with synthetic peptides for generation of antibodies. The peptides were coupled to keyhole limpet hemocyanin (KLH) via a N- or C-terminal cysteine residue. The following peptide sequences were used: humanMDH2.sup.preseq MLSALARPASAALRRSFST-Cys (SEQ ID No.: 11), corresponding to presequence amino acids 1-19; yeastSod2.sup.preseq MFAKTAAANLTKKGED-Cys (SEQ ID No.: 12), corresponding to presequence amino acids 1-16; yeastCox4.sup.preseq MLSLRQSIRFFKPATRT-Cys (SEQ ID No.: 13), corresponding to presequence amino acids 1-17 and humanVDAC3 Cys-GKNFSAGGHKVGLGFELEA (SEQ ID No.: 14).
(108) Mouse and human proteins analysed by immunodecoration were probed with the following primary antibodies: anti-Amyloid- (The Genetics Company, WO-02), anti-Cytochrome c (BD Pharmingen), mtHSP70 (anti-GRP75, Abcam, ab2799), SDHA (Abcam, ab14715).
(109) SDS-PAGE and Immunodecoration was performed according to standard protocols and developed using ECL Western Blotting Detection Reagents (GE Healthcare) and X-ray films or the LAS 4000 system (Fujifilm). Non-relevant lanes were excised by digital processing.
(110) Cox4.sup.preseq (MLSLRQSIRFFKPATRTLCSSRYLL) (SEQ ID No.: 15), Sdh1.sup.octa (FTSSALVR; EZ Biolab) (SEQ ID No.: 16) and A.sup.1-28 (DAEFRHDSGYEVHHQKLVFFAEDVGSNK; Anaspec) (SEQ ID No.: 17) peptides were dissolved in dH.sub.2O to a stock concentration of 1 mM and stored at 20 C. A.sup.1-40 peptides (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV; Sigma, A1075) (SEQ ID No.: 18) were dissolved in dH.sub.2O to a stock concentration of 500 M, sonicated (two times 10 s with 10 s breaks) and stored at 80 C. A.sup.scrambl. peptides (KVKGLIDGDHIGDLVYEFMDSNSAIFREGVGAGHVHVAQVEF)(SEQ ID No.: 19) were dissolved in 0.1% (w/v) NH.sub.4OH to a stock concentration of 1 mM and stored at 20 C. For in vitro processing assays the stocks were further diluted in reaction buffer.
(111) TABLE-US-00003 TABLE S1 Yeast strains used in this study. # Name Genotype Reference 1501 Wild-type MATa; ade2-101; his3-200; leu2-1; ura3- (40) 52; trp1-63; lys2-801 3372 cym1 MATa; ade2-101; his3-200; leu2-1; ura3- This study 52; trp1-63; lys2-801; YDR430c::TRP1 3675 oct1 MATa; ade2-101; his3-200; leu2-1; ura3- This study 52; trp1-63; lys2-801; YKL134c::HIS3MX6 3676 oct1cym1 MATa; ade2-101; his3-200; leu2-1; ura3- This study 52; trp1-63; lys2-801; YDR430c::TRP1; YKL134c::HIS3MX6 2263 mas1 Mat; ura3-52; trp1-1; leu2-3; (41) leu2-112; his3-11; his3-15 3508 mas1cym1 Mat; ura3-52; trp1-1; leu2-3; This study leu2-112; his3-11; his3- 15; YDR430c::HIS3MX6 2876 Cym1.sup.H84Y MAT; can1100; his311, 15; leu23, 112; (26) ura31; ade21; trp11; YDR430c::KanMX6; YCplac111- CYM1(H84Y) 2874 Cym1.sup.H88Y MAT; can1100; his311, 15; leu23, 112; (26) ura31; ade21; trp11; YDR430c::KanMX6; YCplac111- CYM1(H88Y) 2873 Cym1.sup.E87Q MAT; can1100; his311, 15; leu23, 112; (26) ura31; ade21; trp11; YDR430c::KanMX6; YCplac111- CYM1(E87Q) 2875 Cym1 MAT; can1100; his311, 15; leu23, 112; (26) ura31; ade21; trp11; YDR430c::KanMX6; YCplac111- CYM1(WT) 3941 coa6 MATa; ade2-101; his3-200; leu2-1; ura3- This study 52; trp1-63; lys2-801; YMR244c-a:: KanMX6
(112) TABLE-US-00004 TABLE S2 Overview of the human brain samples used in this study: Sample Registration# Age (y) Sex Post mortem (h) C1 184 91 Female 12 C2 190 86 Female 14 C3 178 79 Female 5 C4 188 64 Female 5 AD1 216 80 Female 12 AD2 253 70 Female 12 AD3 215 72 Female 8 AD4 255 78 Female 12
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