FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis

AbstractHuntington's disease is characterised by the death of cortical and striatal neurons, and is the result of an expanded polyglutamine tract in the Huntingtin protein [1]. Huntingtin is present on both endocytic and secretory membrane organelles but its function is unclear [2,3]. Rab GTPases regulate both of these transport pathways [4]. We have previously shown that Rab8 controls polarised membrane transport by modulating cell morphogenesis [5]. To understand Rab8-mediated processes, we searched for Rab8-interacting proteins by the yeast two-hybrid system. Here, we report that Huntingtin is linked to the Rab8 protein through FIP-2, a tumour necrosis factor-α (TNF-α)-inducible coiled-coil protein related to the NEMO protein [6,7]. The activated form of Rab8 interacted with the amino-terminal region of FIP-2, whereas dominant-negative Rab8 did not. Huntingtin bound to the carboxy-terminal region of FIP-2. Coexpressed FIP-2 and Huntingtin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures, and FIP-2 promoted cell polarisation in a similar way to Rab8. We propose a model in which Huntingtin, together with FIP-2 and Rab8, are part of a protein network that regulates membrane trafficking and cellular morphogenesis

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.Rab8 reglerar cellens form och utseende. En avhandling vid medicinska fakulteten, Helsingfors universitet beskriver hur det GTP bindande proteinet Rab8 reglerar cellens form och utseende (cellpolarisering). I en flercellig organism måste celler specialicera sig och röra på sig för att organismen skall utvecklas korrekt. För att detta skall kunna ske måste de först ändra form i en process kallad polarisering. Cellens komponenter organiseras då assymetriskt för att bilda funktionellt skilda delar. Att en cell kan röra på sig har inte bara en essentiell funktion under utvecklingsskedet, det är också en process som behövs under hela organismens livstid. Det behövs t.ex. rörliga celler för att bekämpa infektioner och för att sår skall kunna läkas. Det är också viktigt att celler som inte skall röra på sig förblir stilla. Strikt kontroll av vilka celler som skall och inte skall röra på sig behövs kontinuerligt. Ett exempel på vad som kan hända om denna kontroll inte fungerar är cancer. Små GTPaser är en stor grupp proteiner som fungerar som omkopplare eller strömbrytare i vidareföring av signaler inuti cellen. Deras funktioner i cellen är diversa och inkluderar bland annat kontroll av celltillväxt, celldelning, och membrantransport. Denna stora grupp (fler än 100 proteiner) kan delas upp i fem familjer: Ras, Rho, Rab, Arf och Ran. Ras var den första humana onkogenen att beskrivas 1981, och i uppskattningsvis en tredjedel av all cancer finns mutationer som leder till ett permanent aktiverat Ras-protein. Rab-familjen av små GTPaser kontrollerar membrantransport i cellen och det är en medlem av denna familj, Rab8, som är fokuset av Katarina Hattulas avhandling. Tidigare forskning har inte klart kunnat visa vilken roll Rab8 har i cellen. I avhandlingen ges bevis på att Rab8 kraftigt påverkar cellens form och utseende. Resultatet av den forskning som presenteras visar en roll för Rab8 i återvinning av material som behövs för att en cell skall kunna bilda och upprätthålla en polariserad fenotyp. Något som bland annat behövs för att cellen skall kunna röra på sig. Avhandlingen presenterar också i detalj hur man sökt efter proteiner som specifikt kan binda till och påverka Rab8s funktion. Tre nya Rab8 bindande protein beskrivs. Ett av dessa är optineurin, som på annat håll har visats vara knutet till glaukom, (en av de främsta anledningarna till blindhet i världen med 33 miljoner drabbade). En koppling mellan optineurin och Huntingtin, det protein vars gen är muterad i personer med Huntingtons sjukdom visas också. Ett annat Rab8 bindande protein som beskrivs är Rabin8. Det aktiverar Rab8 och behövs för att Rab8 ska kunna utföra sin uppgift vid cellpolartisering. Sammantaget ger den forskning som presenteras en ny insikt i hur membrantransport reglerad av Rab8 aktivt bidrar till att ändra cellens form och utseende. Detta kan ge en ny insikt i cellulära processer som cellmigration såväl som en rad sjukdomsförlopp till exempel glaukom, Huntingtons sjukdom och cancer

Association between the Intrinsically Disordered Protein PEX19 and PEX3

Peer reviewe

The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells

The full length human PEX3 protein sequence.

<p>A) The red letters indicate amino acids which are not expressed in the recombinant protein. The black letters indicate amino acids modelled in the X-ray structure (PDB ID: 3AJB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101-Sato1" target="_blank">[40]</a>. The blue letters indicate amino acids which are not modelled in the X-ray structure. B) X-ray structure of PEX3 with a PEX19 peptide bound (PDB ID: 3AJB). The secondary structure of PEX3 is colored as a) helix (orange), b) sheet (dark blue) and c) coil (pink). The PEX19 peptide is shown in turquoise.</p

Comparison of hydrogen exchange in PEX19 alone and in complex with PEX3 monitored by MS.

<p>(<b>A</b>) HXMS heat map of PEX19 monomer summarizing the deuterium uptake over time. (<b>B</b>). HXMS heat map of PEX19 in the heterodimer with PEX3 summarizing the deuterium uptake over time. (<b>A and B</b>). The sequence of the protein is shown above the heat map. Although the recombinant protein includes an N-terminal tag only the full sequence of human PEX19 is shown with the numbering starting at the initial PEX19 methionine. The heat maps were assembled from individual peptic peptides using MSTools <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101-Kavan1" target="_blank">[47]</a>. The extent of the peptide is demarcated by vertical lines in each block, running through all four time point bars, when there is a difference in uptake from the preceding or following peptide. All peptic peptides are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101.s001" target="_blank">Figure S1</a></b>. The scale bar at the bottom of each heat map illustrates the color coding for deuterium uptake as a percentage. The horizontal bars from the top to the bottom in each block of the heat map indicate incubation times of 0, 3, 7, 40 and 60 minutes, respectively. White bars represent the residues for which no data were available.</p

The heat map of PEX3 plotted on the X-ray structure of PEX3 with a PEX19 peptide bound (PDB ID: 3AJB) [40].

<p>Regions where the hydrogen/deuterium exchange decreased in the heterodimer compared to PEX3 alone are shown in red. Regions where the exchange remained the same are shown in blue. The left hand panel is rotated 180° along the Y axis compared to the right hand panel.</p

The full length human PEX3 protein sequence.

<p>A) The red letters indicate amino acids which are not expressed in the recombinant protein. The black letters indicate amino acids modelled in the X-ray structure (PDB ID: 3AJB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103101#pone.0103101-Sato1" target="_blank">[40]</a>. The blue letters indicate amino acids which are not modelled in the X-ray structure. B) X-ray structure of PEX3 with a PEX19 peptide bound (PDB ID: 3AJB). The secondary structure of PEX3 is colored as a) helix (orange), b) sheet (dark blue) and c) coil (pink). The PEX19 peptide is shown in turquoise.</p