MARCO CONCEPTUAL SEPSIS NEONATAL A. INTRODUCCIÓN

Very rarely during surgery the temperature of a patient suddenly starts to rise uncontrollably. Even when heroic measures are taken, sudden death may follow within minutes. This malignant hyperthermia syndrome is often associated with administration of halogenated anesthetics such as a widely used mixture of halothane (2-bromo-2-chloro- 1,1,1-trifluoroethane) and succinylcholine.a– d _There

is often no warning that the patient is abnormally sensitive to anesthetic. However, development of an antidote together with increased alertness to the problem has greatly decreased the death rate. Nevertheless, severe damage to nerves and kidneys may still occur.c _{Biochemical investigation of the}

hyperthermia syndrome has been facilitated by the discovery of a similar condition that is prevalent among certain breeds of pigs. Such “stress-prone” pigs are likely to die suddenly of hyperthermia induced by some stress such as shipment to market. The sharp rise in temperature with muscles going into a state of rigor is accompanied by a dramatic lowering of the ATP content of the muscles.

The problem, both in pigs and in humans susceptible to malignant hyperthermia, was found in the Ca2+ _{release channels (ryanodine receptors).}

Study of inheritance in human families together

a _{Gordon, R. A., Britt, B. A., and Kalow, W., eds. (1973) Interna-}

tional Symposium on Malignant Hyperthermia, Thomas, Spring-

field, Illinois

b _{Clark, M. G., Williams, C. H., Pfeifer, W. F., Bloxham, D. P.,}

Holland, P. C., Taylor, C. A., and Lardy, H. A. (1973) Nature

(London) 245, 99 – 101

c _{MacLennan, D. H., and Phillips, M. S. (1992) Science 256, 789 – 794}

d _{Simon, H. B. (1993) N. Engl. J. Med. 329, 483 – 487}

e _{Fujii, J., Otsu, K., Zorzato, F., de Leon, S., Khanna, V. K., Weiler,}

J. E., O’Brien, P. J., and MacLennan, D. H. (1991) Science 253, 448 – 451

f _{MacLennan, D. H., Duff, C., Zorzato, F., Fujii, J., Phillips, M.,}

Korneluk, R. G., Frodis, W., Britt, B. A., and Worton, R. G. (1990) Nature (London) 343, 559 – 561

g _{Sakube, Y., Ando, H., and Kagawa, H. (1997) J. Mol. Biol. 267,}

849 – 864

with genetic studies in pigs led to the finding that the stress-prone pigs have cysteine replacing argin- ine 615 in the Ca2+ _{channel protein. This modifica-}

tion appears to facilitate opening of the channels but to inhibit their closing.e _{A similar mutation has}

been found in some human families in which the condition has been recognized. However, there is probably more than one site of mutation in humans.c,f

Similar mutations in the nematode C. elegans are being investigated with the hope of shedding light both on the problem of hyperthermia and on the functioning of the Ca2+ _{release channels.}g

sensor is revealed by a lethal mutation (muscular disgenesis) in mice. Animals with this autosomal recessive trait generate normal action potentials in the sarcolemma but Ca2+ _{is not released and no muscular}

contraction occurs. They lack a 170-kDa dihydro- pyridine-binding subunit of the sensor.246

Some aspects of regulation by calcium ions are poorly understood. The frequent observations of oscillations in [Ca2+_{] in cells is described in Box 6-D.}

Another phenomenom is the observation of Ca2+

“sparks,” detected with fluorescent dyes and observa- tion by confocal microscopy.247 _{These small puffs of}

Ca2+ _{have been seen in cardiac muscle}247 _{and in a}

somewhat different form in smooth muscle.248 _They

may represent the release of Ca2+ _{from a single release}

channel or a small cluster of channels. When the calcium release channels open, Ca2+ _{ions flow from the cisternae}

of the SR into the cytoplasm, where they activate both the troponin–tropomyosin system and also the Ca2+_-

calmodulin-dependent light chain protein kinase, which acts on the light chains of the myosin head. These light chains resemble calmodulin in their Ca2+_-

binding properties. The function of light chain phos- phorylation of skeletal muscle myosin is uncertain but it is very important in smooth muscle.248a

The regulatory complex of tropomyosin and tro- ponin is attached to the actin filaments as indicated in Fig. 19-8D and also in Fig. 19-9. The latter shows a model at near atomic resolution but without side chains on the tropomyosin and without the troponin components. When the regulatory proteins are com- pletely removed from the fibrils, contraction occurs until the ATP is exhausted. However, in the presence of the regulatory proteins and in the absence of calcium, both contraction and hydrolysis of ATP are blocked. Tropomyosin (Tm) is a helical coiled-coil dimer, a 40-nm rigid rod, in which the two 284-residue 33-kDa mono- mers have a parallel orientation (Fig. 19-9)249 _{as in the}

myosin tail. However, an 8 – to 9– residue overlap at the ends may permit end-to-end association of the Tm molecules bound to the actin filament. As with other muscle proteins there are several isoforms,250,251 _whose

distribution differs in skeletal and smooth muscle and in platelets. The elongated Tm rods appear to fit into the grooves between the two strands of actin mono- mers in the actin filament.252– 254 _{In resting muscle the}

Tm is thought to bind to actin near the site at which the S1 portion of the myosin binds. As a consequence, the Tm rod may block the attachment of the myosin heads to actin and prevent actin-stimulated hydrolysis of ATP. The 40-nm Tm rod can contact about seven actin subunits at once (Fig. 19-9). Thus, one Tm–troponin complex controls seven actin subunits synchronously.

Troponin (Tn) consists of three polypeptides (TnC, TnI, TnT) that range in mass from 18 to 37 kDa. The complex binds both to Tm and to actin.255,256 _Peptide

TnT binds tightly to Tm and is thought to link the

TnI

·

TnC complex to Tm.256,257 _{TnI interacts with}

actin and inhibits ATPase activity in the absence of Ca2+_.258– 261 _{It may work with the other two peptides}

to keep the Tm in the proper position to inhibit ATP hydrolysis. TnC binds calcium ions. This ~160-residue protein has a folding pattern almost identical to that of calmodulin (Fig. 6-8) with four Ca2+_{-binding domains}

arranged in two pairs at the ends of a long 9-turn helix. When Ca2+ _{binds to TnC, a conformational change}

occurs258,259,262-265 _{(p. 313). This induces changes in}

the troponin

·

tropomyosin

·

actin complex, releases the inhibition of actomyosin ATPase, and allows contrac- tion to occur.265a _{In the heart additional effects are}

exerted by β-adrenergic stimulation, which induces phosphorylation of two sites on TnI by the action of the cAMP-dependent protein kinase PKA. Dephos- phorylation by protein phosphatase 2A completes a regulatory cycle in which the doubly phosphorylated TnI has a decreased sensitivity to calcium ions.266

Cardiac muscle also contains a specialized protein called phospholamban. An oligomer of 52-residue subunits, it controls the calcium ion pump in response toβ-adrenergic stimulation. Unphosphorylated phos- pholamban inhibits the Ca ATPase, keeping [Ca2+_]

high in the cytoplasm. Phosphorylation of phospho- lamban by cAMP and/or calmodulin-dependent protein kinase activates the Ca2+ _pump,267–268a _{removing Ca}2+

and ending contraction.

X-ray diffraction and electron microscopy in the 1970s suggested that when calcium binds to troponin the tropomyosin moves through an angle of ~ 20° away from S1, uncovering the active site for the myosin– ATP–actin interactions.252,253 _{Tropomyosin could be}

envisioned as rolling along the surface of the actin, uncovering sites on seven actin molecules at once. Side-chain knobs protruding from the tropomyosin like teeth on a submicroscopic gear might engage complementary holes in the actin. At the same time a set of magnesium ion bridges between zones of nega- tive charge on tropomyosin and actin could hold the two proteins together. This proposal has been difficult to test. Although the older image reconstruction is regarded as unreliable, recent work still supports this steric blocking model.255,269-270c _{Image reconstruction}

from electron micrographs of thin filaments shows that, in the presence of Ca2+_{, the tropomyosin does}

move 25° away from the position in low [Ca2+_{]. How-}

ever, instead of two states of the thin filament, “on” and “off,” there may be at least three, which have been called “blocked,” “closed,” and “open.”255,269,271,271a

The closed state may be attained in rigor.269 _{In addi-}

tion, the possibility that changes in the conformation of actin as well as of myosin occur during the contrac- tion cycle must be considered.255

Smooth muscle. The primary regulation of smooth muscle contraction occurs via phosphorylation of the Ser 19 – OH group in the 20-kDa regulatory light chains of each myosin head.121b,160,272– 274 _{The phosphorylated}

form is active, participating in the contraction process. Removal of Ca2+ _{by the calcium pump and dephospho-}

rylation of the light chains by a protein phosphatase275

restores the muscle to a resting state. The N-terminal part of the myosin light chain kinase binds to actin, while the catalytic domain is in the center of the pro- tein. The C-terminal part binds to myosin, and this binding also has an activating effect.276

Another protein, caldesmon, binds to smooth muscle actin and blocks actomyosin ATPase.271,277–278a

It is present in smooth muscle in a ratio of actin:tropo- myosin:caldesmon of ~14:2:1. Inhibition can be reversed by Ca2+_{, but there is no agreement on the function of}

caldesmon.277 _{It is an elongated ~ 756-residue protein}

with N-terminal domain, which binds to myosin, and a C-terminal domain, which binds to actin, separated by a long helix.278 _{Caldesmon may be a substitute for}

troponin in a tropomyosin-type regulatory system, or it may promote actomyosin assembly. Another possi- bility is that it functions in a latch state, an energy- economic state of smooth muscle at low levels of ATP hydrolysis.278,279 _{In molluscan muscles Ca}2+ _{binds to a}

myosin light chain and activates contraction directly. Some molluscan smooth muscles (catch muscles) also have a latch state, which enables these animals to maintain muscular tension for long periods of time, e.g., holding their shells tightly closed, with little expenditure of energy.279a _{Catch muscles contain}

myosin plus a second protein, catchin, which is formed as a result of alternative mRNA splicing. Catchin contains an N-terminal sequence that may undergo phosphorylation as part of a regulatory mechanism.280 _{However, recent experiments indicate}

that twitchin (see next paragraph), rather than catchin, is essential to the catch state and is regulated by phos- phorylation.280a _{Regulation of the large groups of}

unconventional myosins is poorly understood. Phos- phorylation of groups on the myosin heavy chains is involved in ameba myosins and others.281

An unexpected aspect of regulation was discovered from study of the 40 or more genes of Caenorhabditis

elegans needed for assembly and function of muscle.

The mutants designated unc-22 showed a constant twitch arising from the muscles in the nematode’s body. The gene was cloned using transposon tagging (Chapter 27) and was found to encode a mammoth 753-kDa 6839-residue protein which has been named twitchin.282– 285 _{Twitchin resembles titin (Fig. 19-8) and}

like titin has a protein kinase domain, which is nor- mally inhibited by the end of its peptide chain, which folds over the active site of the kinase. Perhaps the protein kinase activities of twitchin, titin, and related proteins285 _{are required in assembly of the sarcomere.}

5. The Phosphagens

ATP provides the immediate source of energy for muscles but its concentration is only ~ 5 mM. As dis- cussed in Chapter 6, Section D, phosphagens, such as creatine phosphate, are also present and may

attain a concentration of 20 mM in mammalian mus- cle. This provides a reserve of high-energy phospho groups and keeps the adenylate system buffered at a high phosphorylation state ratio286 _{(see Eq. 6-67).}

The concentration of both ATP and creatine phos- phate as well as their rates of interconversion can be monitored by 31_{P NMR within living muscles (Figs.}

6-4 and 19-22). Phospho groups were observed to be Creatine-P + ADP Creatine + ATP

Figure 19-22 Phosphorus-31 magnetic resonance spectra of

wrist flexor muscles of the forearm of a trained long-distance runner at rest and during contraction at three different levels of exercise. Ergometer measurements indicating the percent of initial maximum strength (% max) were recorded over each 6-min period. Spectra were obtained during the last 3 min of each period. Times of spectral data collection: A, resting; B, 4–6 min; C, 10–12 min; and D, 16–18 min. The pH

ranged from 6.9 to 7.0. From Park et al.288

20 10 0 -10 Chemical shift (ppm) Resting 20% 40% 60% maximum effort Pi PCr β α γ ATP A B C D

transferred from creatine phosphate to ADP to form ATP with a flux of 13 mmol kg–1_s–1 _{in rat legs.}287 _The

reverse reaction must occur at about the same rate because little cleavage of ATP to P_i was observed in the anaesthetized rats. Use of surface coils has permitted direct observation of the operation of this shuttle system in human muscle (Fig. 19-22)288 _{as well as in}

animal hearts (see Chapter 6). Only a fraction of the total creatine present within cells participates in the shuttle, however.289

C. Motion in Nonmuscle Cells

At one time actin and myosin were thought to be

present only in muscles, but we know now that both actin proteins of the myosin family are present in all eukaryotic cells. Ameboid movement, the motion of cilia and flagella, and movement of materials along microtubules within cells also depend upon proteins of this group.

1. Actin-Based Motility

Ameboid movements of protozoa and of cells from higher organisms, the ruffling movements of cell membranes, phagocystosis, and the cytoplasmic streaming characteristic of many plant cells289a _have

all been traced to actin filaments or actin cables rather

Bind and stabilize Profilina,b,c,d,e,f,g,h

monomeric actin ADF/Cofilini,j,h,k

Thymosinf,l

Cap actin filament ends CapZm,n

barbed end Fragmino,p

pointed end β-Actinq

Tropomodulinr

Arp2/3, a complex

of seven polypeptides

Sever or dissociate Gelsolins,t,u,v,w

actin filament Depactin

Profilind,e

ADF/cofilinh,k,x,y

TABLE 19-1

Some Actin-Binding Proteins

Function Name

a_{Aderem, A. (1992) Trends Biochem. Sci. 17, 438 – 443} b _{Mannherz, H. G. (1992) J. Biol. Chem. 267, 11661– 11664} c _{Way, M., and Weeds, A. (1990) Nature (London) 344, 292 – 293} d _{Eads, J. C., Mahoney, N. M., Vorobiev, S., Bresnick, A. R., Wen, K.-K.,}

Rubenstein, P. A., Haarer, B. K., and Almo, S. C. (1998) Biochemistry 37, 11171– 11181

e_{Vinson, V. K., De La Cruz, E. M., Higgs, H. N., and Pollard, T. D. (1998)}

Biochemistry 37, 10871– 10880

f _{Kang, F., Purich, D. L., and Southwick, F. S. (1999) J. Biol. Chem. 274, 36963 –} 36972

g _{Gutsche-Perelroizen, I., Lepault, J., Ott, A., and Carlier, M.-F. (1999) J. Biol.}

Chem. 274, 6234 – 6243

h _{Nodelman, I. M., Bowman, G. D., Lindberg, U., and Schutt, C. E. (1999) J.}

Mol. Biol. 294, 1271– 1285

i _{Lappalainen, P., Fedorov, E. V., Fedorov, A. A., Almo, S. C., and Drubin, D.} G. (1997) EMBO J. 16, 5520 – 5530

j_{Rosenblatt, J., and Mitchison, T. J. (1998) Nature (London) 393, 739 – 740} k _{Chen, H., Bernstein, B. W., and Bamburg, J. R. (2000) Trends Biochem. Sci. 25,}

19 – 23

l _{Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, M. L.,}

Vandekerckhove, J., Perelroizen, I., Yin, H., Doi, Y., and Pantaloni, D. (1996)

J. Biol. Chem. 271, 9231– 9239

m _{Barron-Casella, E. A., Torres, M. A., Scherer, S. W., Heng, H. H. Q., Tsui, L.-} C., and Casella, J. F. (1995) J. Biol. Chem. 270, 21472 – 21479

n _{Kuhlman, P. A., and Fowler, V. M. (1997) Biochemistry 36, 13461– 13472} o _{Steinbacher, S., Hof, P., Eichinger, L., Schleicher, M., Gettemans, J.,}

Vandekerckhove, J., Huber, R., and Benz, J. (1999) EMBO J. 18, 2923 – 2929 p _{Khaitlina, S., and Hinssen, H. (1997) Biophys. J. 73, 929 – 937}

q_{See main text}

r_{Gregorio, C. C., Weber, A., Bondad, M., Pennise, C. R., and Fowler, V. M.} (1995) Nature (London) 377, 83 – 86

s_{Azuma, T., Witke, W., Stossel, T. P., Hartwig, J. H., and Kwiatkowski, D. J.} (1998) EMBO J. 17, 1362 – 1370

t_{De Corte, V., Demol, H., Goethals, M., Van Damme, J., Gettemans, J., and} Vandekerckhove, J. (1999) Protein Sci. 8, 234 – 241

u _{McGough, A., Chiu, W., and Way, M. (1998) Biophys. J. 74, 764 – 772} v_{Sun, H. Q., Yamamoto, M., Mejillano, M., and Yin, H. L. (1999) J. Biol. Chem.}

274, 33179 – 33182

w_{Robinson, R. C., Mejillano, M., Le, V. P., Burtnick, L. D., Yin, H. L., and} Choe, S. (1999) Science 286, 1939 – 1942

x_{Carlier, M.-F., Ressad, F., and Pantaloni, D. (1999) J. Biol. Chem. 274, 33827–} 33830

y_{McGough, A., and Chiu, W. (1999) J. Mol. Biol. 291, 513 – 519}

z_{Markus, M. A., Matsudaira, P., and Wagner, G. (1997) Protein Sci. 6, 1197–} 1209

aa_{Vardar, D., Buckley, D. A., Frank, B. S., and McKnight, C. J. (1999) J. Mol.}

Biol. 294, 1299 – 1310

bb_{Matsudaira, P. (1991) Trends Biochem. Sci. 16, 87– 92}

cc_{Ono, S., Yamakita, Y., Yamashiro, S., Matsudaira, P. T., Gnarra, J. R.,} Obinata, T., and Matsumura, F. (1997) J. Biol. Chem. 272, 2527– 2533 dd_{McLachlan, A. D., Stewart, M., Hynes, R. O., and Rees, D. J. G. (1994) J.}

Mol. Biol. 235, 1278 – 1290

ee_{Tsukita, S., Yonemura, S., and Tsukita, S. (1997) Trends Biochem. Sci. 22, 53 –} 58

ff_{Tsukita, S., and Yonemura, S. (1999) J. Biol. Chem. 274, 34507– 34510}

Crosslink actin filaments or monomers

Tight bundles Villinc,z,aa

Loose bundles α-Actininbb

Spectrinbb

Fascincc

MARCKSa

Network Filaminbb,c

Gelactins

Bind actin filaments Talindd

to membrane “ERM” proteinsee,ff

than to microtubules.289b _{Actin is one of the most}

abundant proteins in all eukaryotes. Its network of filaments is especially dense in the lamellipodia of cell edges, in microvilli, and in the specialized stereocilia and acrosomal processes (see also pp. 369-370).289c

Actin filaments and cables are often formed rapidly and dissolve quickly. When actin filaments grow, the monomeric subunits with bound ATP are added most rapidly at the “barbed end” and dissociate from the filament at the “pointed end” (see Section B,2).94,290

The rate of growth may be ~ 20 – 200 nm / s, which requires the addition of 10 – 100 subunits / s.291 _Various

actin-binding proteins control the growth and stability of the filaments. The actin-related proteins Arp2 and Arp3, as a complex Arp2/3, together with recently recognized formins.291a_{, provide nuclei for rapid}

growth of new actin filaments as branches near the barbed ends.290,292–293c

Growth of the barbed ends of actin filaments is stimulated by phosphoinositides and by members of the Rho family of G proteins (p. 559)293d _{through inter-}

action with proteins of the WASp group.293b,d,e _The

name WAS comes from the immune deficiency disorder Wiskott–Aldrich Syndrome. Yet another family, the Ena/VASP proteins, are also implicated in actin dy- namics. They tend to localize at focal adhesions and edges of lamellipodia.293e,f _{Profilin (Table 19-1) stabiliz-}

es a pool of monomeric actin when the barbed ends of actin filaments are capped. However, it catalyzes both the addition of actin monomers to uncapped barbed ends and rapid dissociation of subunits from pointed ends, leading to increased treadmilling.294,295 _Actin-

severing proteins such as the actin depolymerizing factor (ADF or cofilin, Table 19-1) promote break- down of the filaments.296–297a _{Treadmilling in the}

actin filaments of the lamellipods of crawling cells or pseudopods of amebas provides a motive force for many cells291,298–299 _{ranging from those of Dictyostelium}

to human leukocytes. A series of proteins known as the ezrin, radixin, moesin (ERM) group attach actin to integral membrane proteins (Fig. 8-17)292,300,301 _and

may interact directly with membrane lipids.301a,b

Bound ATP in the actin subunits is essential for poly- merization, and excess ATP together with crosslinking proteins stabilize the filaments. However, when the bound ATP near the pointed ends is hydrolyzed to ADP the filaments become unstable and treadmilling is enhanced. Thus, as in skeletal muscle, ATP provides the energy for movement.

Bacteria also contain filamentous proteins that resemble F-actin and which may be utilized for cell- shape determination.301c _{Actin-based motility is used}

by some bacteria and other pathogens during invasion of host cells (Box 19-C). It is employed by sperm cells of Ascaris and of C. elegans, which crawl by an ameboid movement that utilizes treadmilling of filaments formed from a motile sperm protein, which does not

resemble actin.302,302a _{Cells are propelled on a glass}

surface at rates up to ~1 µm / s.

Various nonmuscle forms of myosin also interact with actin without formation of the myofibrils of muscle.299 _{In most higher organisms nonmuscle myo-}

sins often consist of two ~ 200-kDa subunits plus two pairs of light chains of ~ 17 and 24 kDa each. These may form bipolar aggregates, which may bind to pairs of actin filaments to cause relative movement of two parts of a cell.303 _{Movement depending upon the}

cytoskeleton is complicated by the presence of a bewil- dering array of actin-binding proteins, some of which are listed in Table 19-1.

2. Transport along Microtubules by Kinesin and Dynein

Many materials are carried out from the cell bodies of neurons along microtubules in the axons, which in the human body may be as long as 1 m. The rates of this fast axonal transport in neurons may be as high as 5 µm / s or 0.43 m / day. The system depends upon ATP and kinesin (Fig. 19-17) and permits small vesi- cles to be moved along single microtubules.304–305b

Movement is from the minus end toward the plus end of the microtubule as defined in Figs. 7-33, 7-34. Slow- er retrograde axonal transport carries vesicles from the synapses at the ends of the axons (Fig. 30-8) back toward the cell body. This retrograde transport depends upon the complex motor molecule cytoplasmic dynein which moves materials from the plus end of the micro- tubule toward the minus end.305,305c _{In addition to}

these movements, as mentioned in Chapter 7, micro- tubules often grow in length rapidly or dissociate into their tubulin subunits. Growth occurs at one end by addition of tubulin subunits with their bound GTP. The fast growing plus-ends of the microtubules are usually oriented toward the cell periphery, while the

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