Small Sticks of Bi­ol­ogy

by Elio

How a pro­teins folds into a par­tic­u­lar shape is about as cen­tral a con­cern as there is in our postge­nomic world. Pro­tein mol­e­cules bend, curl up, writhe, and, gen­er­ally un­dergo big time changes in shape and form, plus they can make strong con­nec­tions be­tween seg­ments along their length and with other mol­e­cules, large and small. Con­cepts such as the sec­ond ge­netic code, Anfinsen's dogma, Levinthal's para­dox, molten glob­ule state, chap­er­ons, dis­or­der, and who knows how many more, all con­spire to ei­ther com­pli­cate or clar­ify (de­pend­ing on out­look) our un­der­stand­ing of how such things work in the liv­ing world. But with­out un­der­stand­ing how pro­teins work we would be left with the sim­pler con­tor­tions of the nu­cleic acids.

Fig­ure 1. A stick. Source

Among the most in­trigu­ing par­tic­i­pants in these mol­e­c­u­lar bal­lets are the amy­loid pro­teins. These are in­sol­u­ble fi­brous mol­e­cules that ag­gre­gate in a spe­cific man­ner dif­fer­ing in sev­eral ways from the "na­tive mol­e­cules". What most dis­tin­guishes them is that they make in­sol­u­ble fib­rils. These changes can have colos­sal con­se­quences, as in the case of the pri­ons, the self-per­pet­u­at­ing amy­loid pro­teins that cause dev­as­tat­ing dis­eases in hu­mans and an­i­mals. But not all is bleak in the world of amy­loids. Mind you, this is a big sub­ject with ram­i­fi­ca­tions ga­lore.

The term amy­loid has been mis­lead­ing from the get-go: it comes from the Latin amy­lum for starch. It was wrongly called that be­cause one of the fa­thers of mod­ern pathol­ogy, Rudolph Vir­chow found in 1854 that ac­cu­mu­la­tions of amy­loid pro­teins in tis­sues be­came stained by io­dine, which is typ­i­cal of starch. But the name stuck. In time, some three dozen patho­log­i­cal con­di­tions have been as­so­ci­ated with the de­po­si­tion of in­sol­u­ble amy­loid fibers, in­clud­ing such well-known dis­eases as Alzheimer's, Parkinson's, mad cow dis­ease, cer­tain type 2 di­a­betes, mul­ti­ple myeloma, plus a num­ber of less known but no less dam­ag­ing ones.

Dis­ease-caus­ing amy­loid pro­teins are formed by pro­te­ol­y­sis of any of a large num­ber of dif­fer­ent pre­cur­sor mol­e­cules that in­clude, among many oth­ers, the im­munoglob­u­lin light chains. The rea­son for the pathol­ogy is that amy­loid pro­teins adopt a hy­dropho­bic cross β‑sheet con­fig­u­ra­tion that leads them to ag­gre­gate into tis­sue-dis­rupt­ing fib­rils. For an­thro­pocen­tric rea­sons, the mol­e­cules in this con­fig­u­ra­tion are called ab­nor­mal or mis­folded (who's to say what's nor­mal or what's folded the "right" way?).

Fig­ure 2. Schematic rep­re­sen­ta­tion of amy­loid poly­mer­iza­tion. (a) Ki­net­ics of sol­u­ble CsgA monomers poly­mer­iz­ing into curli (flu­o­res­cence is from bind­ing the amy­loid bind­ing dye thioflavin T).  Blue ar­row: end of the lag phase. In­sert: trans­mis­sion EM of CsgA fibers formed in vitro. Scale bar, 500 nm. (b) The tran­si­tion from sol­u­ble monomeric pro­teins to poly­meric and in­sol­u­ble amy­loid fibers is in steps that re­sult in loss- or gain-of-func­tion prop­er­ties for the pro­tein. In the lag phase, sol­u­ble pro­tein as­sem­bles into a com­mon in­ter­me­di­ate or nu­cleus that is pro­posed to be toxic to mem­branes. The for­ma­tion of the in­ter­me­di­ate is pro­posed to be the rate-lim­it­ing step of amy­loido­ge­n­e­sis. Once the nu­cleus is formed, monomers are tem­plated into grow­ing amy­loid fibers. When the monomer pop­u­la­tion is de­pleted, elon­ga­tion stops and en­ters the sta­tion­ary phase. The green boxes be­low the schematic high­light prop­er­ties of some of the func­tional amy­loids and the red boxes some gen­eral prop­er­ties of dis­ease-as­so­ci­ated amy­loids. Source

Amy­loid Re­demp­tion

Amy­loid pro­teins may sure sound dis­rep­utable, but that's grossly un­fair be­cause many of them play ben­e­fi­cial roles. Among these is par­tic­i­pat­ing in the for­ma­tion of long term-mem­ory, caus­ing an alarm re­ac­tion to virus in­fec­tion, and pos­si­bly even ame­lio­rat­ing symp­toms in mul­ti­ple scle­ro­sis and other neu­roin­flam­ma­tory con­di­tions. But it is in bac­te­ria and fungi that the good side of amy­loid pro­teins is eas­i­est to find. Here, amy­loids are in­volved in such dis­parate ac­tiv­i­ties as mak­ing ad­he­sive struc­tures ("curli" in E. coli and other Gram neg­a­tives), form­ing biofilms (where they are of­ten the ma­jor pro­tein com­po­nents), or help­ing in the con­struct of aer­ial struc­tures (chap­lins) in mold-like actin­o­mycetes. And there is more. Amy­loids make up the pili that fa­cil­i­tate at­tach­ment of tu­ber­cle bacilli to host cells and the ad­hesins of Can­dida al­bi­cans. And amy­loids are the ben­e­fi­cial pri­ons in yeast. For a fine re­view of amy­loids as the good guys, click here.

But even among mi­cro­bial amy­loids, there are some that to re­vert to their orig­i­nal rep­u­ta­tion.  Kleb­siella pneu­mo­niae, for in­stance, makes a bac­te­ri­ocin, Mi­crocin E492, a pep­tide that as­sem­bles as a tran­sient oligomeric in­ter­me­di­ate in the cell mem­brane of other bac­te­ria with dire con­se­quences. But in ad­di­tion, this pro­tein causes apop­to­sis in some hu­man cell lines, bring­ing up ques­tions about the con­nec­tion be­tween mi­cro­bial "func­tional" amy­loids and dis­ease caus­ing ones.

The Play­ers in Amy­loido­ge­n­e­sis

Now that we are back in our mi­cro­bi­o­log­i­cal home (at least for many of the read­ers of this blog), we can turn to a spe­cific study on the role of amy­loids, in this case re­gard­ing the for­ma­tion of a pro­tein of B. sub­tilis that is es­sen­tial for biofilm for­ma­tion. Like with other bac­te­ria, for mak­ing a biofilm B. sub­tilis must make an ex­tra­cel­lu­lar ma­trix that binds the cells to­gether. In this species, the net­work is com­posed of both a poly­sac­cha­ride and an amy­loid pro­tein. This pro­tein is called TasA and is in­vari­ably ac­com­pa­nied by a small amount of an­other pro­tein, BslA that makes a thin hy­dropho­bic film. This story in­volves a trio of genes in an operon that en­odes the ma­jor amy­loid pro­tein, TasA; a mi­nor com­po­nent, TapA; and SipW, a busy pro­tein that func­tions as a pep­ti­dase to processes the other two and also reg­u­lates ex­opolysac­cha­ride pro­duc­tion. In TapA null mu­tants, TasA fibers don't as­so­ciate with the cell sur­face but dis­perse in the medium. Thus, TapA is an aux­il­iary pro­tein that is re­quired for fib­ril for­ma­tion. How does it do it? And why do we want to know? The an­swer lies in the com­plex­i­ties of amy­loid fiber for­ma­tion. This is not a sim­ple as­sem­bly of sub­units into a poly­mer. Rather, ac­ces­sory pro­teins con­trol the cor­rect as­sem­bly of fibers and the process goes through a num­ber of de­fined steps.

Fig­ure 3. Dele­tion of TapA residues 50 to 57 in­hibits biofilm for­ma­tion. Top row: Pel­li­cle for­ma­tion of tapA wild type and mu­tant strains. Amylo: tapA of B. amyloli­quefa­ciens. re­place: strain with re­place­ment of amino acids 50–57. Bot­tom row: Colony mor­phol­ogy of the same strains. Source

To visit for a mo­ment to the bet­ter-known sys­tem of curli fibers, here also two struc­tural pro­teins are in­volved. The mi­nor one (CsgB) con­tributes to the poly­mer­iza­tion of the ma­jor one (CsgA). Pos­si­bly, CsgB pro­vides a tem­plate for the cor­rect fold­ing of CsgA. Some­thing like this has been pro­posed for the in­ter­ac­tion of TasA and TapA.  The re­searchers, from the Losick and Kolter labs, used an in vitro as­say that al­lows to mea­sure how the ad­di­tion of TapA en­hances the poly­mer­iza­tion of TasA. They de­fined a re­gion of 8 amino acids in TapA that is re­quired for func­tion, pos­si­bly by par­tic­i­pat­ing in the tran­si­tion of TasA from monomer to poly­mer and in mak­ing fibers on the sur­face of the cell. These are nice steps on the way to un­der­stand how amy­loid fibers are made and in elu­ci­dat­ing their im­por­tant roles. This sub­ject promises to fur­ther oc­cupy cen­ter stage in mi­cro­bi­ol­ogy as it has else­where.

How to Make Amy­loids

I fin­ish with a quote from a fine re­view by Chap­man and col­lab­o­ra­tors:

"…. the bad rap of amy­loids is rapidly bright­en­ing. For starters, ma­ture amy­loid fibers are prob­a­bly not the cause of Alzheimer's dis­ease. An even more im­por­tant fac­tor in re­shap­ing the im­age of amy­loid has been the dis­cov­ery of func­tional amy­loids, which have taught us that the amy­loid fold can be an im­por­tant part of cel­lu­lar phys­i­ol­ogy. As our ap­pre­ci­a­tion for amy­loid bi­ol­ogy con­tin­ues to grow, it will be im­por­tant to fully vet the mech­a­nisms that cel­lu­lar sys­tems have evolved to as­sem­ble, dis­as­sem­ble and con­trol amy­loid for­ma­tion. The ma­ture amy­loid fold found in fib­rils does not ap­pear to be in­her­ently toxic if han­dled ap­pro­pri­ately. How­ever, sim­i­lar to many things, amy­loid for­ma­tion at the wrong time, in the wrong place or with the wrong con­stituents can be cat­a­strophic. The func­tional amy­loids pro­vide a how-to guide for co­or­di­nat­ing or­dered pro­tein ag­gre­ga­tion in ways such that other cel­lu­lar processes are not neg­a­tively im­pacted."

 

Ref­er­ence

Romero D, Vla­makis H, Losick R, Kolter R (2014). Func­tional analy­sis of the ac­ces­sory pro­tein TapA in Bacil­lus sub­tilis amy­loid fiber as­sem­bly. Jour­nal of Bac­te­ri­ol­ogy, 196 (8), 1505–13 PMID: 24488317