Section A (25 marks in total)

A.1)     Read the paper below, and write a max. 100 word abstract and provide a title.

Functional deterioration and accumulation of damage to the proteome is an inevitable consequence of    cellular aging. This damage is largely repaired through protein turnover where potentially impaired           polypeptides are replaced with new, functional copies. These turnover mechanisms are particularly          important in post-mitotic cells, such as neurons, because they cannot dilute potentially toxic species        through cell division. Nearly all proteins within the human proteome are recycled in less than a few days. However, a few extremely long-lived proteins (ELLPs) with half-lives on the order of months have been   identified, including myelin basic protein (MBP). Additional ELLPs probably remain to be discovered. For example, a subset of nuclear pore complex (NPC) proteins, which form transport channels responsible    for mediating nuclear trafficking, are present but no longer expressed in differentiated cells. Thus at least a subset of nucleoporins (Nups), are not, or are only very slowly, replaced during adulthood. However,    because worms have a life span of a few weeks, it remains unclear if NPC components remain incorpo-  rated in the nuclear membrane over years, particularly in the central nervous system of mammals, which contain non-dividing cells that are as old as the organism itself. To explore this question, we performed   pulse chase labeling of whole rats with the stable isotope 15N followed by mass spectrometry to monitor global protein turnover on a timescale of years (the average life span of a lab rat is 2 years). Two female Sprague Dawley rats and their progeny were fed a 15N-enriched algal cell diet, and at 6 weeks all           progeny rats were switched to a 14N diet. Fully 15N labelled (t=0) rats were immediately sacrificed and   their tissues harvested. Nuclei from liver, an organ that turns over within 4-6 months, and brain were        purified, digested with trypsin, and analyzed by MudPIT (multidimensional protein identification techno-   logy) LCLC-MS/MS (multidimensional liquid chromatography-tandem mass spectrometry). At time=0, we calculated 15N isotopic protein labelling efficiency of >98% and identified more than 3,400 fully 15N        proteins (20,754 peptides) and only 9 14N proteins (14 peptides). Two additional animals were sacrificed at 6 and 12 months and 15N/14N ratios were determined for more than 3,500 unique proteins. Only 7      heavy (15N) proteins (11 peptides) were found in the liver after 6 months, consistent with the relatively    rapid turn-over of hepatocytes. In contrast, the brain contained a large number of heavy peptides (92       peptides) even after 12 months (Fig. 1B, fig. S1E). These peptides corresponded to 25 proteins and        included MBP and histones, the latter having reported half-lives of ~220 days in mouse brain (8),             confirming the validity of our approach (Fig. 1A). All the other heavy proteins identified were components of the two essential core modules of the NPC, the pentameric Nup205 complex and the nonameric          Nup107- 160 complex (Fig. 1B and Table S1). This represents an essential intracellular protein machine  with protein components in excess of a year in age. Detailed analysis of 15N spectral counts and             15N/14N MS1 ratios revealed that in contrast to the stable scaffold, the peripheral Nups and components of the central transport channel were devoid of heavy peptides, suggesting they were completely replaced after 6 months (Fig. 1C). Thus, unlike other large protein complexes, such as the proteasome    or ribosome, in which all components have similar turnover values, the individual components of NPCs    have very different lifetimes. This supports the idea that NPCs are built to last the entire lifespan of the    cell and are not completely removed and assembled anew in post-mitotic cells. Rather, NPC                    maintenance in non-dividing cells relies on the non- or extremely slow- exchange of scaffold and rapid     replacement of peripheral Nups. A lack of protein turn-over exposes the proteome to an increased risk of aberrant chemical modifications and oxidative damage during aging. Indeed, healthy rats exhibit age-      dependent decline of NPC function. Our results suggest that ELLPs represent a diverse class of proteins that regulate essential cellular functions and could be linked directly to the decline of the aging proteome.

Fig. 1. Identification of NUPs and histones as extremely long-lived proteins in mammalian brain. (A) MS1       scans (indicated M/Z ranges) at 0 and 6 months. Distinct peptides for indicated proteins; red indicates 15N    peptide peaks, black 14N, grey other peptides, and asterisk peaks were successfully identified by MS/MS.     (B) Distribution of 15N MS/MS spectral counts (485) grouped as proteins (25) from 12 month brain nuclei and 6 month liver nuclei (inset); histones (black), NUPs (red/light red), and MBP (green). (C) Relative MS1 peak   quantitation for each Nup with heavy peptide hits indicated as 15N /14N ratios when possible.

Section B (50 marks in total)

FRET studies of Sarcolipin-SERCA interactions

Sarcolipin (SLN) is a 3 kDa membrane protein that reversibly inhibits the activity of the calcium-transporting ATPase (SERCA) in sarcoplasmic reticulum (SR) of fast-twitch, slow-twitch, and atrial muscle. SERCA is a 110-kDa membrane protein that relaxes muscle by pumping calcium out of the cytoplasm using energy derived from ATP hydrolysis. SLN gene expression is up-regulated 2– 15-fold in patients with skeletal muscle dysferlinopathy and Takotsubo cardiomyopathy, but down-regulated 2–3-fold in patients with heart failure, atrial fibrillation, and congenital heart defects.

SLN comprises a single transmembrane (TM) helix, plus a small cytoplasmic phosphorylation domain and a short lumenal tail. SLN is monomeric on SDS-PAGE but aggregates when purified in non-ionic detergent, suggesting that SLN may oligomerise in SR membranes. Despite the evidence demonstrating that SLN regulation of SERCA is a major determinant of muscle contractility, the physiological role and oligomeric structure of the SLN channel are unknown.

To investigate the oligomeric state of SLN, CFP-SLN and YFP-SLN were separately and simultaneously over expressed in Sf21 insect cells. In this case, CFP (cyan fluorescence protein) is the donor fluorophore and YFP (yellow fluorescence protein) is the acceptor fluorophore. Cell homogenates were analysed by flourescence spectroscopy. The detergent sodium dodecyl sulphate (SDS) was added to homogenates to disrupt any SLN-SLN interactions.

B.1) Briefly describe the principle of FRET (Förster resonance energy transfer) and how it can be used to investigate molecular structure. Use max. 4 bullet points. (8 marks)

CFP-SLN is excited at 420nm and emits light at 480 nm.

YFP-SLN is excited at 480nm and emits light at 520 nm

B.2) Why is light emitted at a different wavelength than excitation? (4 marks)

Table 1: FRET studies of SLN oligomerisation in cell homogenates. The excitation wavelength used is given above each set of experiments.

FRET efficiency (FRETeff) is defined as:        FRETeff = 1 - (FDA/FD)

where FDA  is the fluorescence of the donor in the presence of acceptor and FD  is the fluorescence of the donor in the absence of acceptor.

B.3) Calculate the FRET efficiency for experiments 5 and 6 in table 1. You may assume that CFP- SLN and YFP-SLN are expressed at the same level whether expressed individually or together. (6 marks)

B.4) Comment on all the data in Table 1 and your calculated FRET efficiencies. What do these data reveal about the oligomeric state of SLN? (5 marks)

A helical wheel representation of the transmembrane region of SLN was used to identify the possible surfaces where SLN could interact with SERCA.

Residues I17, L16 and I22 of SLN were mutated to alanine. FRET efficiency assays using cell homo- genates coexpressing YFP-SLN and CFP-SERCA were used to probe the effect of these mutations on the formation of the SLN:SERCA complex.

Table 2: FRET studies of YFP-SLN binding to CFP-SERCA.

B.5) Comment on the data in Table 2. Which face of the transmembrane helix (a, b, c, d, e, f or g) is likely to be at the interface between SLN and SERCA? (3 marks)

Figure 1: To scale structural cartoons of the fluorescent protein fusions of SLN and SERCA. The CFP or YFP portions are highlighted by enclosure in a dotted line.

B.6) With reference to Figure 1, your answer to part (a) and the data presented in Table 2, suggest possible reasons for the difference in FRET efficiency observed for the CFP-SLN:YFP-SLN and YFP-SLN:CFP-SERCA complexes. (6 marks)

SLN is known to undergo phosphorylation of its small cytoplasmic domain. In order to determine whether SLN  phospohrylation  plays  a  role  in  regulating  the  SLN:SERCA  interaction.  Cell  homogenates coexpressing CFP-SERCA and YFP-SLN, as well as homogenates coexpressing CFP-SERCA and a constituively phosphorylated YFP-SLN (designated at YFP-SLN*) were assayed by FRET.

By progressively photobleaching YFP the effective concentration in the cell homogenates can be reduced in  a  controlled  manner.  This  enables  the  measurement  of  FRET  efficiency  as  a  function  of  the concentration of acceptor fluorophore (YFP-SLN or YFP-SLN*).

[YFP-SLN]

(uM)

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

[YFP-SLN*]

(uM)

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

FRET efficiency is related to [acceptor] by the following relationship:

B.7) Use equation 1 and a suitable graphical approach to determine the Kd and FRETmax values for the YFP-SLN:CFP-SERCA and YFP-SLN*:CFP-SERCA interactions. (12 marks)

B.8) Deduce from your answer to (e) whether phosphorylation promotes or reduces the binding of SLN and SERCA and explain your answer. (2 marks)

Many viruses encode small transmembrane proteins with one or two membrane spanning helices that oligomerise to form pores in the host cell membrane that increase membrane permeability.

Phospholamban, another SERCA regulatory protein with similar structure to SLN has been shown to have poor forming activity. In order to investigate whether SLN is able to form higher-order oligomers that could contain a central pore cell viability assays were carried out.

E. coli cells were transfected with his-tagged SLN, I17A SLN, chloramphenical transferase (as a non-pore- forming negative control) and phospholamban (as a positive control). A plating assay was used to measure cell viability. All proteins were expressed at similar concentrations within the cell.

140

120

100

80

60

40

20

0

CON                  PLB                  SLN                  I17A